Oligonucleotide encoded chemical libraries

ABSTRACT

This application provides a bead with a covalently attached chemical compound and a covalently attached DNA barcode and methods for using such beads. The bead has many substantially identical copies of the chemical compound and many substantially identical copies of the DNA barcode. The compound consists of one or more chemical monomers, where the DNA barcode takes the form of barcode modules, where each module corresponds to and allows identification of a corresponding chemical monomer. The nucleic acid barcode can have a concatenated structure or an orthogonal structure. Provided are method for sequencing the bead-bound nucleic acid barcode, for cleaving the compound from the bead, and for assessing biological activity of the released compound.

CROSS REFERENCE TO RELATED CASES

This application claims the benefit of, and priority to, U.S.Provisional Patent Application Ser. No. 62/562,905 filed Sep. 25, 2017,and U.S. Provisional Patent Application Ser. No. 62/562,912, also filedSep. 25, 2017, the contents of which are incorporated herein byreference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure relates to high-throughput screening using a library ofcompounds, where the compounds are bound to beads, or contained withinbeads, each bead containing multiple copies of one kind of compound,where further, the bead also contains DNA tags that encode the identityor synthetic history of the compound that is contained in or on thebead. The disclosure also relates to high-throughput assays performed inpicowells, where the picowells contain compound-laden beads and assaymaterials. The disclosure further relates to releasing the bead-boundcompounds and screening them for biological activity. Broadly, thedisclosure contemplates assays where beads are used as delivery-vehiclesfor compounds, and methods for creating such compound-laden beads.

The disclosure relates bead-bound compounds, where each compound is madeof one or more monomers belonging to a chemical library. The disclosurealso relates to bead-bound DNA barcodes, that is, to nucleic acids wherethe sequence of each nucleic acid is a code (not related to the geneticcode) refers to one particular chemical library monomer. The disclosurefurther relates to releasing the bead-bound compounds and then screeningthe released compounds for biological activity.

The disclosure also pertains generally to methods for perturbing a cell,or a few cells, with a dose-controlled compound, and analyzing thechange in the state of the cell by RNA and/or protein analysis. Themethods disclosed herein could be applied at the single-cell level, orto a plurality of cells, for the purpose of high throughput screening,target discovery, or diagnostics, and other similar applications.

BACKGROUND OF THE DISCLOSURE

Combinatorial chemistry, for example, involving split-and-poolchemistry, can be used for synthesizing large amounts of compounds.Compounds made in this way find use in the field of medicinal chemistry,where the compounds can be screened for various biochemical activities.These activities include binding to one or more proteins, where theproteins are known at the time the screening test is performed.Alternatively, the proteins that are bound by a compound being testedare identified only after a binding event is detected. Compounds canalso be screened for their activity of inhibiting or activating a knownprotein (this is not merely screening for a “binding” activity).Alternatively, compounds can be screened for their activity ofinhibiting or activating a cellular function, and where the moleculartargets are not known to the researcher at the time of screening.

The screening of compounds, such as compounds belonging to a hugelibrary of chemicals made by split-and-pool methods, can be facilitatedby conducting screening with an array of many thousands of microwells,nanowells, or picowells. Moreover, screening can be facilitated byproviding a different compound to each picowell by way of a bead, andwhere each bead contains hundreds of copies of the same compound, andwhere the same bead also contains hundreds of copies of a “DNA barcode”that can be used to identify the compound that is attached to the samebead. Moreover, screening of compounds is further facilitated by usingcleavable linkers, where the cleavable linker permits controlled releaseof the compound from the bead, and where the released compound is thenused for biochemical assays or cell-based assays in the same picowell.

Assaying compounds in very small, confined volumes, such as droplets,picowells or microfluidic environments is broadly beneficial, forinstance, due to the low volumes of assay reagents needed, and thereforeneed not be limited to combinatorially generated compounds. Any methodthat can load compounds onto beads, that also allows the compounds to beeluted off the beads at a later time, may be used for deliveringbead-bound compounds to assays in small, confined volumes. The additionof nucleic acid barcodes to the beads allows the identity of thecompound present within the beads to be carried along to the assayvolume. In his manner, very high throughput assays may be performedwithout needing robotics or spatial indexing of compounds withinmicrotiter plates. Millions to billions of compounds may be held withinone small vial, the identity of the compounds tagged on the same bead(with DNA) that contains each individual compound.

A common method for drug discovery involves picking a target of interestand monitoring the interaction of the target protein or enzyme with alarge library of chemical compounds. In many cases, a large number ofinitial hits are found toxic to the body or cross reactive with otherproteins in the body, rendering the target-based selection aninefficient method for drug screening. The need for a pre-selectedtarget is also an inherent limitation, since it requires the biologicalunderpinning of disease to be well-known and understood. Screeningcompounds against an entire organism is a difficult, expensive, and verylow-throughput task.

Conventional phenotypic screening on cells has involved creating modelsof diseased-state cells, contacting the cells with various druglibraries, and monitoring if the disease phenotype is corrected by ameasurable assay. Such screening methods are called phenotypicscreening, as the underlying biological mechanism is not necessarilyunderstood at the beginning, but a measurable, phenotypic change that isindicative of a curative response is considered the relevant metric. Avast number of cell lines and disease models reflecting various baselineand diseased cell states are available today. Also available are largernumbers of compound libraries and biological drugs candidates. Theobvious screening campaign combining different cell models withdifferent drug candidates to look for phenotypic responses is fraughtwith technical limitations as assays are limited to microtiter plateformats and imaging modalities, both of which are severely limited inthroughput.

One method to overcome throughput limitations is to adopthigh-throughput single-cell screening approaches to drug discovery (see,e.g., Heath et al., Nat Rev Drug Discov. 15:204-216, 2016). In theseapproaches, single cells are separated and isolated into compartmentswhere individual assays can be performed on each of the cells. Genomicanalysis via mRNA sequencing of the single cells, e.g., using dropletencapsulation, is a popular method that reveals intricate details thatare hidden in ensemble measurements (see, e.g., Macosko et al., Cell161:1202-1214, 2015 and Ziegenhain et al., Mol Cell 65:631-643, 2017,the disclosures of which are incorporated herein by reference in theirentireties). Present state of the art single-cell analysis platformshave enabled quantitation of mRNA transcripts with single-cellresolution to characterize and fingerprint cells based on theirtranscriptional state. This approach allows for comparison betweentissue samples, extracted from a subject or prepared in an experiment,and examining single-cell transcription, and therefore, proteinexpression states. The measurements of single-cell mRNA by transcriptomesequencing and profiling are important approaches to investigatemolecular mechanisms of not only genealogic phenotypes of cells duringdisease progression, but also drug efficacy, resistances, and discoveryof therapeutic targets (see, e.g., Chu et al., Cell Biol and Toxicol33:83-97, 2017, Wang, Cell Biol Toxicol 32:359-361, 2016, and Wang etal., Cell Biol Toxicol 33:423-427, 2017). The application of single-cellRNA sequencing has been used to define intercellular heterogeneity,evidenced by transcriptomic cell-to-cell variation, which is extremelyrelevant to drug efficacy and specificity, transcriptionalstochasticity, transcriptome plasticity, and genome evolution.Encapsulation in picowells has also been demonstrated (see, e.g.,Gierahn et al., Nat Methods 14:395-398, 2017). Single cell proteinmeasurements are also possible using similar isolation methods (Butniket al., BioRxiv, January 2017, Su et al., Proteomics 17:3-4, 2017).

Despite the rapid rise in high-throughput single-cell RNA-sequencing(RNA-seq) methods, including commercialized versions of automatedplatforms such as the Fluidigm C1, 10×Genomics or 1CellBiO systems, theapplication of single-cell RNA profiling for target agnostichigh-throughput drug screening and target discovery is constrained bythe lack of methods that can efficiently partition different drugs todifferent cells. While incubating cells or tissues under differentperturbations within well plates, followed by single-cell analysis andcomparisons between transcript profiles can be done, the number of drugsthat can be examined is limited by the plate capacity. Further, the needto prepare barcoded mRNA from each sample in isolation and then performcomprehensive RNA profiles for every sample, creates a major bottleneck,as well.

SUMMARY OF THE DISCLOSURE

Briefly stated, the present disclosure provides a system for screeningchemical compounds, comprising: (a) A picowell array plate comprising aplurality of picowells, wherein each picowell has a top aperture thatdefines an opening at the top of the picowell, a bottom that is definedby a floor, wherein the top aperture is separated from the floor, andwherein a wall resides in between the top aperture and the floor; (b) Abead disposed in a picowell, wherein the bead comprises a plurality ofsubstantially identical bead-bound DNA barcodes, and a plurality ofsubstantially identical bead-bound compounds, (c) Wherein the beadcomprises a bead-bound DNA barcode that takes the form of either aconcatenated DNA barcode or an orthogonal DNA barcode, and wherein ifthe DNA barcode takes the form of a concatenated DNA barcode theconcatenated DNA barcode is made by a method that: (i) Uses clickchemistry, or (ii) Uses a repeating cycle of steps, wherein therepeating cycle of steps comprises using a splint oligonucleotide(splint oligo) that is capable of hybridizing to a partially madebead-bound DNA barcode, and wherein the hybridizing is mediated by anannealing site on the splint oligo and a corresponding, complementaryannealing site in the partially made bead-bound DNA barcode, wherein theannealed splint oligo is used as a template for extending the partiallymade DNA barcode using DNA polymerase, and wherein the splint oligocontains bases that are complementary to a DNA barcode module that is tobe polymerized to the partially made DNA barcode, (d) Wherein each oneof the plurality of substantially identical bead-bound compoundscomprises one or more chemical library monomers, and wherein eachbead-bound DNA barcode module identifies a corresponding chemicallibrary monomer, wherein the term “compound” is used to refer to acompleted product that comprises one or more chemical library members,and wherein the completed DNA barcode identifies the compound.

The floor of a microwell, nanowell, or picowell, need not be flat. Thefloor may be curved as in the manner of the bottom of a glass test tubeor metal centrifuge tube. Also, the floor may be conical-shaped, as inconical centrifuge tubes. The floor may be flat but with notches, forexample, notches that facilitate motion of an assay solution or cellculture solution in the vicinity of the bottom of any bead that issitting in the picowell. In flat-floor embodiments, the present systemand methods can require a flat floor.

The concatenated DNA barcode can be made entirely by methods of organicchemistry, for example, by click chemistry. Also, the orthogonal DNAbarcode can be made entirely by methods of organic chemistry, forexample, comprising click chemistry.

What is also provided is the above system, further comprising aplurality of caps, each cap capable of fitting into the opening of adifferent picowell, and each cap capable of minimizing or preventingevaporation of fluid that is inside of the picowell, and each capable ofminimizing or preventing leakage of fluid that is inside of thepicowell.

Moreover, what is embraced is the above system, wherein the concatenatedDNA barcode is made by a method that uses: (i) Both click chemistry andthe repeating cycle of steps that uses the splint oligo; (ii) Both clickchemistry and chemical methods that are not click chemistry methods;(iii) Only click chemistry; or (iv) Only the repeating cycle of stepsthat uses the splint oligo. For this particular embodiment the“concatenated DNA barcode” in question does not include any chemicalcoupler that is used to couple a nucleic acid directly to the bead.

In a spherical cap embodiment, what is provided is the above system,further comprising a plurality of spherical caps, wherein each cap iscapable of fitting into the aperture of a picowell wherein the apertureis circular, and each cap is capable of minimizing or preventingevaporation of fluid that is inside of the picowell, and each cap iscapable of minimizing or preventing leakage of fluid that is inside ofthe picowell.

In a response element embodiment, what is provided is the above system,wherein the at least one bead disposed in the at least one picowellcomprises at least one response capture element that is coupled to saidat least one bead. Also, what is contemplated is the above system,wherein the at least one bead disposed in at least one picowellcomprises at least one response capture element that is coupled to saidat least one bead, wherein the at least one response capture elementcomprises: (a) Poly(dT) or (b) An exon-targeting RNA probe.

Also contemplated is the above system, wherein the DNA barcode is eithera concatenated DNA barcode or an orthogonal DNA barcode, and wherein theDNA barcode comprises one or more DNA barcode modules, wherein each ofthe one or more DNA barcode modules encodes information that identifiesa chemical library monomer, and wherein the concatenated DNA barcode orthe orthogonal DNA barcode further includes one or both of: (a) One ormore functional nucleic acids; and (b) One or more nucleic acids thatencode information of a type other than the identity of a chemicallibrary monomer.

The following discloses “consists of only” embodiments and “comprises”embodiments, as it applies to the number of bead-bound DNA barcodemodules that make up a DNA barcode. What is provided is embodimentswhere the DNA barcode consists of only one DNA barcode module, or onlytwo DNA barcode modules, or contains only three DNA barcode modules, oronly four DNA barcode modules, and so on, or where the DNA barcodecomprises at least one DNA barcode module, or comprises at least two DNAbarcode modules, or comprises at least three DNA barcode modules, orcomprises at least four DNA barcode modules, and so on,

What is also embraced, is a system wherein the bead-bound concatenatedDNA barcode comprises: (i) a 1^(st) DNA barcode module; or (i) a 1^(st)DNA barcode module, a 1^(st) annealing site, and a 2^(nd) DNA barcodemodule; or (ii) a 1^(st) DNA barcode module, a 1^(st) annealing site, a2^(nd) DNA barcode module, a 2^(nd) annealing site, and a 3^(rd) DNAbarcode module; or (iii) a 1^(st) DNA barcode module, a 1^(st) annealingsite, a 2^(nd) DNA barcode module, a 2^(nd) annealing site, a 3^(rd) DNAbarcode module, a 3^(rd) annealing site, and a 4^(th) DNA barcodemodule; or (iv) a 1^(st) DNA barcode module, a 1^(st) annealing site, a2^(nd) DNA barcode module, a 2^(nd) annealing site, a 3^(rd) DNA barcodemodule, a 3^(rd) annealing site, a 4^(th) DNA barcode module, a 4^(th)annealing site, and a 5^(th) DNA barcode module; or (v) a 1^(st) DNAbarcode module, a 1^(st) annealing site, a 2^(nd) DNA barcode module, a2^(nd) annealing site, a 3^(rd) DNA barcode module, a 3^(rd) annealingsite, a 4^(th) DNA barcode module, a 4^(th) annealing site, a 5^(th) DNAbarcode module, a 5^(th) annealing site, and a 6^(th) DNA barcodemodule.

Moreover, what is contemplated is the above system, further comprising aprimer binding site capable of binding a DNA sequencing primer, whereinsaid primer binding site is capable of directing sequencing of one ormore of the 1 t DNA barcode module, the 2^(nd) DNA barcode module, the3^(rd) DNA barcode module, the 4^(th) DNA barcode module, the 5^(th) DNAbarcode module, or the 6^(th) DNA barcode module, and wherein the primerbinding site is situated 3-prime to the 1st DNA barcode module, 3-primeto the 2^(nd) DNA barcode module, 3-prime to the 3^(rd) DNA barcodemodule, 3-prime to the 4^(th) DNA barcode module, 3-prime to the 5^(th)DNA barcode module, or 3-prime to the 6^(th) DNA barcode module, orwherein the primer binding site is situated in between the 1^(st) and2^(nd) DNA barcode modules, or is situated in between the 2^(nd) and3^(rd) DNA barcode modules, or is situated in between the 3^(rd) and4^(th) DNA barcode modules, or is situated between the 4^(th) and 5^(th)DNA barcode modules, or is situated between the 5^(th) and 6^(th) DNAbarcode modules.

Additionally, what is provided is the above system, wherein the primerbinding site is situated in between the 1^(st) and 2^(nd) DNA barcodemodules, or is situated in between the 2^(nd) and 3^(rd) DNA barcodemodules, or is situated in between the 3^(rd) and 4^(th) DNA barcodemodules, or is situated between the 4^(th) and 5^(th) DNA barcodemodules, or is situated between the 5^(th) and 6^(th) DNA barcodemodules. In embodiments relating to the position of a primer bindingsite, relative to upstream DNA barcode modules and relative todownstream DNA barcode modules, what is provided is the above system,wherein a primer binding site is situated in between each and every pairof successive DNA barcode modules.

Furthermore, what is provided is the above system, wherein the beadcomprises a DNA barcode that is an orthogonal DNA barcode, wherein thebead comprises an external surface, and wherein the orthogonal DNAbarcode comprises: (a) A first nucleic acid that comprises a first DNAbarcode module and an annealing site for a sequencing primer, whereinthe first nucleic acid is coupled to the bead at a first position, (b) Asecond nucleic acid that comprises a second DNA barcode module and anannealing site for a sequencing primer, wherein the second nucleic acidis coupled to the bead at a second position, and (c) A third nucleicacid that comprises a third DNA barcode module and an annealing site fora sequencing primer, wherein the second nucleic acid is coupled to thebead at a third position, and wherein the first, second, and thirdposition on the bead are each located at different location on thebead's external surface.

In encoding embodiments, what is provided is the above system, whereinthe DNA barcode comprises one or more nucleic acids that do not identifyany chemical library monomer but that instead identify: (a) The class ofchemical compounds that is cleavably attached to the bead; (b) The stepnumber in a multi-step pathway of organic synthesis; (c) The date thatthe bead-bound compound was synthesized; (d) The disease that thebead-bound compound is intended to treat; (e) The cellular event thatthe bead-bound compound is intended to stimulate or inhibit; or (f) Thereaction conditions that were used to couple a given chemical librarymonomer to the bead.

In linker embodiments, what is provided is the above system, whereineach of the plurality of substantially identical bead-bound compounds iscoupled to the bead by way of a cleavable linker. Also provided is theabove system, wherein each of the plurality of substantially identicalbead-bound compounds is coupled to the bead by way of a light-cleavablelinker. Also provided is the above system, wherein each of the pluralityof substantially identical bead-bound compounds is coupled to the beadby way of a non-cleavable linker.

In TentaGel® embodiments, what is provided is the above system, whereinthe at least one bead comprises grafted copolymers consisting of a lowcrosslinked polystyrene matrix on which polyethylene glycol (PEG) isgrafted.

In release-monitor embodiments, the present disclosure provides theabove system, wherein at least one picowell contains a release-monitorbead, and does not contain any other type of bead,

wherein the release-monitor bead comprises a bead-bound quencher and abead-bound fluorophore, wherein the bead-bound quencher is quenchinglypositioned in the immediate vicinity of the bead-bound fluorophore andcapable of quenching at least 50% (or at least 60%, or at least 70%, orat least 80%, or at least 90%, or at least 95%, or at least 99%, or atleast 99.5%, or at least 99.9%) of the fluorescence of the bead-boundfluorophore, and wherein the bead-bound fluorophore is bound by way of afirst light-cleavable linker, wherein the picowell containing therelease-monitor bead is a first picowell, wherein the first picowellcontains a first solution, wherein exposing the first picowell tocleaving conditions is capable of severing the light-cleavable linkerand releasing the fluorophore into the first solution of the firstpicowell, wherein the exposing results in the fluorophore diffusingthroughout the first solution in the first picowell, and wherein afluorescent signal acquired by shining light on the first picowell thatcontains the first solution comprising diffused fluorophore allows theuser to use the fluorescent signal to calculate the percent release ofthe bead-bound fluorophore from the release-monitor bead resulting in avalue for the calculated percent release, and wherein a second picowellcontains a bead-bound compound coupled with the same type oflight-cleavable linker as the first light-cleavable linker, and whereinthe second picowell contains a second solution,

and wherein the value for the calculated percent release from therelease-monitor bead in the first picowell allows calculation of theconcentration of the released compound in the second solution of thesecond picowell.

In embodiments relating to identity of all of the compounds bound to agiven bead, or relating to identity of all of the DNA barcodes bound toa given bead, what is provided is the above system, wherein the at leastone bead comprises a plurality of substantially identical bead-bound DNAbarcodes, wherein the plurality is between 10 million to 100 millioncopies of the substantially identical bead-bound DNA barcodes. Alsoprovided is the above system, wherein the at least one bead comprises aplurality of substantially identical bead-bound compounds, where whereinthe plurality is between 10 million to 100 million copies of thesubstantially identical bead-bound compounds.

In embodiments relating to cells (e.g., mammalian cells, cancer cells,bacterial cells), what is provided is the above system, wherein at leastone picowell comprises at least one cell, wherein the plurality ofsubstantially identical bead-bound compounds are bound to the at leastone bead by way of a cleavable linker, and wherein cleaving thecleavable linker releases the bead-bound compound from the bead toproduce a released compound, and wherein the released compound iscapable of contacting the at least one cell. In other cell embodiments,what is provided is the above system, wherein at least one picowellcomprises at least one cell, wherein the plurality of substantiallyidentical bead-bound compounds are bound to the at least one bead by wayof a cleavable linker, and wherein cleaving the cleavable linkerreleases the bead-bound compound from the bead to produce a releasedcompound, and wherein the released compound is capable of contacting theat least one cell, and wherein the at least one cell is: (i) a mammaliancell that is not a cancer cell, (ii) a mammalian cancer cell, (iii) adead mammalian cell, (iv) an apoptotic mammalian cell, (v) a necroticmammalian cell, (vi) a bacterial cell, (vii) a plasmodium cell, (vii) acell that is metabolically active but has a cross-linked genome and isunable to undergo cell division, or (ix) a mammalian cell that isinfected with a virus.

In device embodiments, what is provided is the above system, whereineach picowell has a top aperture that defines an opening at the top ofthe picowell, a bottom that is defined by a floor, wherein the topaperture is separated from the floor, and wherein a wall resides inbetween the top aperture and the floor, and wherein the aperture isround, wherein the floor is round, and wherein the wall takes the formof a truncated cone, and wherein the aperture has a first diameter, thefloor has a second diameter, and wherein the first diameter is greaterthan the second diameter.

In other device-related embodiments, what is provided is the abovesystem, wherein each picowell has a top aperture that defines an openingat the top of the picowell, a bottom that is defined by a floor, whereinthe top aperture is separated from the floor, and wherein a wall residesin between the top aperture and the floor, and wherein the aperture isround, wherein the floor is round, and wherein the wall takes the formof a truncated cone, and wherein the aperture has a first diameter, thefloor has a second diameter, and wherein the first diameter is greaterthan the second diameter, further comprising a cap that snuggly fitsinto the aperture, wherein the aperture is comprised by a polymer havinga greater durometer (harder) and wherein the cap is made of a polymerhaving a lesser durometer (softer), and wherein the relative durometersof the cap and aperture allow the cap to be reversibly and snuggly fitinto the aperture, and wherein the cap is: (i) a cap intended only toplug the picowell and prevent leakage, (ii) a cap that is a passive capand that is capable of absorbing metabolites that are released by acell, in the situation where a cell in a cell medium is cultured in thepicowell, (iii) a cap that is an active cap, and that takes the form ofa bead that comprises a plurality of essentially identical compounds,and wherein each of the plurality of essentially identical compounds iscoupled to the bead with a cleavable linker; (iv) a cap that is anactive cap, and that takes the form of a bead that comprises a pluralityof identical reagents, and wherein each of the plurality of essentiallyidentical reagents is coupled to the bead with a cleavable linker. Alsoprovided is the above system, wherein the cap is spherical, or whereinthe cap is non-spherical.

In embodiments, the above system comprises a picowell array platecomprising an upper generally planar surface, a plurality of picowells,wherein each picowell has a top aperture that defines an opening at thetop of the picowell, a bottom that is defined by a floor, wherein thetop aperture is separated by a wall from the floor, and wherein the wallresides in between the top aperture and the floor, and optionally, abead disposed in at least one of said plurality of picowells, whereinthe bead comprises a plurality of substantially identical bead-bound DNAbarcodes, and a plurality of substantially identical bead-boundcompounds, wherein the picowell array plate further comprises a mat thatis capable of securely covering the opening at the top of at least oneor all of the plurality of picowells, or that is actually securelycovering the opening at the top of at least one or all of the pluralityof picowells, wherein the securely covering is reversible, wherein themat optionally comprises one or all of: (a) An absorbent surface that,when positioned in contact with the upper generally planar surface ofthe picowell array plate, is capable of absorbing any metabolites,biochemicals, or proteins that may be comprised by one or more of theplurality of picowells, (b) An adhesive surface that is capable ofmaintaining reversible adhesion to the top generally planar surface ofthe picowell array plate.

In biochemical assay embodiments, what is embraced is the above system,that includes at least one picowell, wherein the at least one picowellcomprises a bead that comprises a plurality of substantially identicalcompounds and a plurality of substantially identical barcodes, whereinthe at least one picowell comprises an assay medium that includescereblon E3 ubiquitin ligase, a substrate of cereblon E3 ubiquitinligase such as Ikaros or Aiolos, and wherein the system is capable ofscreening for compounds that activate cereblon's E3 ubiquitin ligaseactivity, and are thereby capable of reducing intracellularconcentrations of Ikaros or Aiolos.

In another biochemical assay embodiment, what is contemplated is theabove system, that includes at least one picowell, wherein the at leastone picowell comprises a bead that comprises a plurality ofsubstantially identical compounds and a plurality of substantiallyidentical barcodes, wherein the at least one picowell comprises an assaymedium that includes MDM2 E3 ubiquitin ligase, a substrate of MDM2 E3ubiquitin ligase such as p53, and wherein the system is capable ofscreening for compounds that activate MDM2's E3 ubiquitin ligaseactivity, and thereby capable of increasing the intracellularconcentrations of p53.

In more barcoding embodiments, what is provided is the above system,wherein the DNA barcode comprises one or more nucleic acids that do notencode any chemical monomer but that instead identify one or more of:(a) The class of chemical compounds that is cleavably attached to thebead; (b) The step in a multi-step pathway of organic synthesis, whereina bead-bound nucleic acid corresponds to a given chemical monomer thatis used to make a bead-bound compound, and wherein the bead-boundnucleic acid that corresponds to a given chemical monomer identifiesthat chemical monomer; (c) The date that the bead-bound compound wassynthesized; (d) The disease that the bead-bound compound is intended totreat; (e) The cellular event that the bead-bound compound is intendedto stimulate or inhibit.

In embodiments that lack any headpiece, what is provided is the abovesystem, wherein the at least one bead comprises a plurality ofsubstantially identical bead-bound compounds and also comprises aplurality of substantially identical bead-bound DNA barcodes, andwherein there does not exist any headpiece that links any of thebead-bound compounds to any of the bead-bound DNA barcodes.

Moreover, what is contemplated is the above system, wherein at least70%, at least 80%, at least 90%, at least 95%, or at least 98% of thesubstantially identical bead-bound DNA barcodes have an identicalstructure. Additionally, what is contemplated is the above system,wherein at least 70%, at least 80%, at least 90%, at least 95%, or atleast 98% of the substantially identical bead-bound compounds have anidentical structure.

Furthermore, what is supplied is the above system, wherein theconcatenated DNA barcode comprises at least one nucleic acid that is aDNA barcode module, or the above system, wherein the concatenated DNAbarcode comprises only one nucleic acid that is a DNA barcode module.

In sequencing primer annealing site embodiments, what is provided is theabove system, wherein the concatenated DNA barcode comprises at leastone nucleic acid that is a DNA barcode module, and at least onefunctional nucleic acid that: (a) Is capable of being used as anannealing site for a sequencing primer, (b) Is capable of forming ahairpin structure, and wherein the hairpin structure comprises asequencing primer, an annealing site for the sequencing primer, and abend in the hairpin structure wherein the bend is 5-prime to thesequencing primer and is 3-prime to the annealing site for thesequencing primer, or (c) Is a spacer nucleic acid.

In other sequencing primer embodiments, what is provided is the abovesystem, wherein the orthogonal DNA barcode contains a plurality of DNAbarcode modules, wherein each of the DNA barcode modules is coupled to adifferent site on the bead either directly or via a linker, and whereineach of the plurality of DNA barcode modules contains at least onefunctional nucleic acid that: (a) Is capable of being used as anannealing site for a sequencing primer, (b) Is capable of forming ahairpin structure, and wherein the hairpin structure comprises asequencing primer, an annealing site for the sequencing primer, and abend in the hairpin structure wherein the bend is 5-prime to thesequencing primer and is 3-prime to the annealing site for thesequencing primer, or (c) Is a spacer nucleic acid.

In embodiments that recite functional language about splint oligos, whatis provided is a bead comprising a concatenated DNA barcode, wherein theconcatenated DNA barcode comprises: (a) a first DNA barcode module and afirst annealing site for a first splint oligonucleotide (splint oligo),wherein the splint oligo comprises three nucleic acids, wherein thethree nucleic acids are: a nucleic acid that is a hybridizing complementto the first annealing site, a nucleic acid that is a hybridizingcomplement to a 2^(nd) DNA barcode module, and a nucleic acid that is a2^(nd) annealing site, and (b) a second DNA barcode module and a 2ndannealing site for a second splint oligo, wherein the second splintoligo comprises three nucleic acids, wherein the three nucleic acidsare: a nucleic acid that is a hybridizing complement to the 2ndannealing site, a nucleic acid that is a 3rd DNA barcode module, and anucleic acid that is a 3rd annealing site.

In another embodiment that contains functional language relating tosplint oligos, what is provided is the above bead, further comprising: athird DNA barcode module and a 3rd annealing site for a third splintoligo, wherein the third splint oligo comprises three nucleic acids,wherein the three nucleic acids are: a nucleic acid that is ahybridizing complement to the 3rd annealing site, a nucleic acid that isa 4^(th) DNA barcode module, and a nucleic acid that is a 4th annealingsite.

Moreover, in yet another embodiment containing functional languagerelating to splint oligos, what is provided is the above bead, furthercomprising one or more of: (i) a fourth DNA barcode module and a 4thannealing site for a fourth splint oligo, wherein the fourth splintoligo comprises three nucleic acids, wherein the three nucleic acidsare: a nucleic acid that is a hybridizing complement to the 4thannealing site, a nucleic acid that is a 5^(th) DNA barcode module, anda nucleic acid that is a 5th annealing site, (ii) a response captureelement, (iii) a release monitor.

In linker embodiments, what is embraced is the above bead, wherein theconcatenated DNA barcode is coupled to the bead, but is: (i) not coupledto the bead by way of any photocleavable linker, (ii) not coupled to thebead by any enzymatically cleavable linker; or (iii) not coupled to thebead by any kind of cleavable linker.

In an embodiment relating to distinct coupling positions, what isprovided is the above bead, wherein the concatenated DNA barcode iscoupled to a first position on the bead, wherein the bead also comprisesa compound that is coupled to a second position on the bead, and whereinthe first position is not the same as the second position.

In surface embodiments (interior and exterior surfaces), what isprovided is the above bead, wherein the bead comprises an exteriorsurface and an interior surface, wherein the bead comprises at least10,000 substantially identical concatenated DNA barcodes that arecoupled to the bead, and wherein at least 90% of the at least 10,000substantially identical concatenated DNA barcodes are coupled to theexterior surface.

In exclusionary embodiments that can distinguish the present disclosurefrom other embodiments, what is provided is the above bead, that is doesnot comprise any polyacrylamide, and wherein the concatenated DNAbarcode: (i) Does not include any nucleic acid that is a promoter; (ii)Does not include any nucleic acid that is polyA; or (iii) Does notinclude any nucleic acid that is a promoter and does not include anynucleic acid that is polyA.

In release-monitor bead embodiments, the present disclosure supplies arelease-monitor bead that is capable of functioning in an aqueousmedium, wherein the release-monitor bead comprises a bead-bound quencherand a bead-bound fluorophore, wherein the bead-bound quencher isquenchingly positioned in the immediate vicinity of the bead-boundfluorophore and capable of quenching at least 50% of the fluorescence ofthe bead-bound fluorophore, and wherein the bead-bound fluorophore isbound by way of a first light-cleavable linker, wherein the picowellcontaining the release-monitor bead is a first picowell, wherein thefirst picowell contains a first solution, wherein exposing the firstpicowell to cleaving conditions is capable of severing thelight-cleavable linker and releasing the fluorophore into the firstsolution of the first picowell, wherein the exposing results in thefluorophore diffusing throughout the first solution in the firstpicowell, and wherein a fluorescent signal acquired by shining light onthe first picowell that contains the first solution comprising diffusedfluorophore allows the user to use the fluorescent signal to calculatethe percent release of the bead-bound fluorophore from therelease-monitor bead resulting in a value for the calculated percentrelease, and wherein a second picowell contains a bead-bound compoundcoupled with the same type of light-cleavable linker as the firstlight-cleavable linker, and wherein the second picowell contains asecond solution, and wherein the value for the calculated percentrelease from the release-monitor bead in the first picowell allowscalculation of the concentration of the released compound in the secondsolution of the second picowell. In other release-monitor embodiments,what is provided is a release-monitor bead wherein the fluorophore isTAMRA and wherein the quencher is QSY7, and a release-monitor bead thathas the structure shown in FIG. 9, and a release-monitor bead of thathas the structure shown in FIG. 10, and a release-monitor bead, whereinthe capable of quenching is at least 90%, at least 98%, at least 99%, orat least 99.9%.

In a methods of manufacture embodiment, what is embraced is a method forsynthesizing a release-monitor bead, wherein the release-monitor beadcomprises a bead, a quencher, a fluorophore, and a photocleavable linkerthat couples the fluorophore to the bead, the method comprising, in thisorder, (i) Providing a resin, (ii) Coupling a lysine linker to theresin, wherein the reagent containing the lysine linker isL-Fmoc-Lys(4-methyltrityl)-OH, (iii) Removing the Fmoc protecting group,(iv) Coupling the quencher using a reagent that isquencher-N-hydroxysuccinimide (quencher-NHS) as the source of quencher,(v) Removing the 4-methyltrityl protecting group using a reagentcomprising trifluoroacetic acid, (vi) Coupling a photocleavable linkerto the epsilon amino group of lysine, wherein the photocleavable linkeris provided by a reagent that is, Fmoc-photocleavable linker-OH, (vii)Coupling the fluorophore. Also provided is the above embodiment, butwithout regard to the ordering of steps. In other methods embodiments,what is provided is the above method wherein the fluorophore is TAMRAand wherein the quencher is QSY7.

In methods relating to the utility of release-monitor bead, what isprovided is a method for controlling the concentration of a compound ina solution that resides in a picowell, wherein the method is applied toa bead-bound compound in a picowell, wherein the picowell contains asolution, and wherein the bead-bound compound is coupled to the bead byway of a cleavable linker, the method comprising: (a) Exposing thebead-bound compound to a condition that effects cleavage of thecleavable linker and releases the bead-bound compound from the bead togenerate a released compound, wherein release is followed by diffusionor dispersion of the released compound in the solution to result in asubstantially uniform concentration of the compound in the solution, (b)Wherein the condition comprises light that is capable of cleaving thecleavable linker, (c) Wherein the condition is adjusted to produce adetermined concentration of the substantially uniform concentration, and(d) Wherein the determined concentration is made with regard to theconcentration of a released fluorophore that is released by from abead-bound release-monitor. Provided also, is the above method, whereinthe condition is adjusted by adjusting one or more of the wavelength ofthe light, the intensity of the light, and by the duration of lightexposure, and the above method, wherein the concentration of a releasedfluorophore that is released from a bead-bound release-monitor isdetermined at the same time as effecting release of the bead-boundcompound from the bead to generate a released compound, and the abovemethod, wherein the concentration of a released fluorophore that isreleased from a bead-bound release-monitor is determined at a timesubstantially before effecting release of the bead-bound compound fromthe bead to generate a released compound.

The term “determined” can mean a concentration that is predetermined anddecided upon as being a desired concentration, prior to exposing thebead to light. Also, the term “determined” can mean a concentration thatis decided upon in “real time,” that is, a concentration that is decidedupon at the same time as the exposing the bead to light.

In cap embodiments, what is embraced is a cap in combination with apicowell plate that comprises a plurality of picowells, wherein the capis capable of use with the picowell plate that comprises a plurality ofpicowells, wherein each of the plurality of picowells is definable by anaperture, a floor, and a wall, wherein the wall is defined by theaperture on top and the floor on the bottom, and wherein the aperture isround, wherein the floor is round, and wherein the wall takes the formof a surface of a truncated cone, and wherein the aperture has a firstdiameter, the floor has a second diameter, and wherein the firstdiameter is greater than the second diameter, wherein the cap is aspherical cap that is capable of snuggly fitting into the aperture,wherein the aperture is comprised by a polymer having a greaterdurometer (harder) and wherein the cap is made of a polymer having alesser durometer (softer), and wherein the relative durometers of thecap and aperture allow the spherical cap to be reversibly and snugglyfit into the aperture, and wherein the cap is: (i) capable of pluggingthe picowell and preventing leakage, (ii) a passive cap and that iscapable of absorbing metabolites that are released by a cell, in thesituation where a cell in a cell medium is cultured in the picowell,(iii) an active cap that takes the form of a bead that comprises aplurality of essentially identical compounds, and wherein each of theplurality of essentially identical compounds is coupled to the bead witha cleavable linker, and wherein cleavage of the cleavable linkerreleases at least some of the plurality of compounds from the bead, (iv)an active cap that takes the form of a bead that comprises a pluralityof identical reagents, and wherein each of the plurality of essentiallyidentical reagents is coupled to the bead with a cleavable linker, andwherein cleavage of the cleavable linker releases at least some of theplurality of reagents from the bead.

In porous cap embodiments, what is provided is a plurality of porouscaps in combination with a picowell plate and a solid polymer coating,wherein each of the plurality of porous caps comprises an upper surfaceand a lower surface, wherein the picowell plate comprises a plurality ofpicowells, wherein at least one porous cap contacts a picowell andreversibly and snuggly fits into the picowell, wherein the picowellplate and each of the upper surfaces of the plurality of porous caps iscovered with a solid polymer coating, wherein the solid polymer coatingcontacts at least some of the upper surface of each cap and isadhesively attached to said at least some of the upper surface, andwherein, (i) Each of the plurality of picowells is capable of holding anaqueous solution, wherein products of a reaction are generated in thesolution, and wherein at least some of the products are absorbed by thelower surface of each of the plurality of porous caps, (ii) Wherein asolution of a polymerizable reagent that capable of polymerization ispoured over the plurality of porous caps in combination with thepicowell plate, and wherein the polymerizable reagent is polymerized toform a substantially planar surface that coats substantially all of thetop surface of the picowell plate, thereby fixing the polymerizedreagent to each of the plurality of porous caps, and (iii) Wherein allof the plurality of porous caps are removable by the act of peeling fromthe plurality of picowells, wherein adhesion is maintained between theplurality of porous caps and the polymerized reagent, resulting in anarray of adhering caps partly with the upper surface of each cap isembedded in the polymerized reagent and the lower surface of each cap isaccessible for analysis of any absorbed reaction product.

This provides a methods of manufacture embodiment, for using splintoligos to guide the enzymatic synthesis of a DNA barcode. What isprovided is a method for making a bead-bound concatenated DNA barcode,wherein the bead-bound concatenated DNA barcode comprises a plurality ofDNA barcode modules, and optionally one or more functional nucleicacids, and optionally one or more identity-encoding nucleic acids thatencode the identity of something other than the identity of a chemicallibrary monomer, the method comprising: (a) The step of providing a beadwith a coupled polynucleotide that comprises a 1^(st) DNA barcode moduleand a 1^(st) annealing site, wherein the 1^(st) annealing site iscapable of hybridizing with a first splint oligonucleotide (splintoligo), the first splint oligo being capable of serving as a templatefor DNA polymerase to catalyze the polymerization to the coupledpolynucleotide, nucleotides that are complementary to those of thehybridized first splint oligo, wherein the polymerized nucleotides thatare complementary to those of the hybridized first splint oligofollowing polymerization comprise a bead-bound 2^(nd) DNA barcode moduleand a 2^(nd) annealing site; (b) The step of providing said bead with acoupled polynucleotide with said first splint oligo, and allowing saidfirst splint oligo to hybridize with said coupled polynucleotide; (c)The step of adding a DNA polymerase and deoxynucleotide triphosphates(dNTPs) and allowing the DNA polymerase to catalyze polymerization ofsaid dNTPs to the coupled polynucleotide, wherein the coupledpolynucleotide has a free 3′-terminus and wherein the polymerization isto the free 3′-terminus, (d) The step of washing away the first splintoligo. Also contemplated is the above method, wherein the first splintoligo comprises a 1^(st) annealing site, a 2^(nd) DNA barcode module,and a 2^(nd) annealing site.

In further methods of manufacture embodiments, what is provided is theabove method, wherein the first splint oligo comprises a 1^(st)annealing site, a 2^(nd) DNA barcode module, a 2^(nd) annealing site,and a nucleic acid encoding a 1^(st) sequencing primer annealing site,wherein the 1^(st) sequencing primer annealing site is capable ofhybridizing to a sequencing primer resulting in a hybridized sequencingprimer, and wherein the hybridized sequencing primer is capable ofdirecting the sequencing of the 2^(nd) DNA barcode module and the 1^(st)DNA barcode module.

Moreover, what is contemplated is the above method, wherein the firstsplint oligo, the DNA polymerase, and the dNTPs are all added at thesame time, or wherein the first splint oligo, the DNA polymerase, andthe dNTPs are each added at separate times.

Regarding interior versus exterior locations on a bead, what is providedis the above method, wherein the bead comprises an exterior location andan interior location, and wherein the bead-bound concatenated DNAbarcode is coupled to the bead at locations that are substantially onthe exterior of the bead and sparingly at interior locations of thebead, and wherein the bead also comprises a plurality of coupledcompounds wherein all of the plurality of coupled compounds havesubstantially an identical structure, when compared to each other, andwherein the bead is comprised substantially of a hydrophobic polymer.

In further methods embodiments, what is provided is the above method,further comprising: (a) The step of providing a bead with a coupledfirst longer polynucleotide that comprises a 1^(st) DNA barcode module,a 1^(st) annealing site, a 2^(nd) DNA barcode, and a 2^(nd) annealingsite, wherein the 2^(nd) annealing site is capable of hybridizing with asecond splint oligo, the second splint oligo being capable of serving asa template for DNA polymerase to catalyze the polymeraztion to thecoupled first longer polynucleotide, nucleotides that are complementaryto those of the hybridized second splint oligo, wherein the polymerizednucleotides that are complementary to those of the hybridized secondsplint oligo following polymerization comprise a bead-bound 3^(rd) DNAbarcode module and a 3^(rd) annealing site; (b) The step of providingsaid bead with a coupled polynucleotide with said 2^(nd) splint oligo,and allowing said 2^(nd) splint oligo to hybridize with said coupledfirst longer polynucleotide; (c) The step of adding a DNA polymerase anddeoxynucleotide triphosphates (dNTPs) and allowing DNA polymerase tocatalyze polymerization of said dNTPs to the coupled longerpolynucleotide, wherein the coupled longer polynucleotide has a free3′-terminus and wherein the polymerization is to the free 3′-terminus,(d) The step of washing away the second splint oligo.

This relates to the consecutive numbering of the first DNA barcodemodule, the second DNA barcode module, the third DNA barcode module, andso on, for the manufacture of the entire DNA barcode. This also relatesto repeating the cycle of methods steps, over and over and over, in themanufacture of the entire DNA barcode. What is provided is the abovemethod, wherein each of said plurality of DNA barcode modules isidentified or named by a number, the method further comprisingreiterating the recited steps, where for a first reiteration, the nameof the DNA barcode module is increased by adding one number to theexisting name, the name of the annealing site is increased by adding onenumber to the existing name, and the name of the splint oligo isincreased by adding one number to the name of the existing distalterminal DNA barcode module, and the name of the “first longerpolynucleotide” is changed by adding one number to the existing name,wherein the comprising reiterating the recited steps is one reiteration,or two reiterations, or three reiterations, or four reiterations, orfive reiterations, or more than five reiterations, or more than tenreiterations.

Also contemplated is the above method, that comprises a plurality ofsplint oligos, wherein each splint oligo comprises a sequencing primerannealing site, wherein the sequencing primer annealing site is capableof hybridizing to a sequencing primer resulting in a hybridizedsequencing primer, and wherein the hybridized sequencing primer iscapable of directing the sequencing of the at least one bead-bound DNAbarcode module and at least one bead-bound DNA barcode module.

This concerns embodiments relating to splint oligos that guides DNApolymerase to synthesize functional nucleic acids and various types ofinformative nucleic acids. What is provided is the above method, whereinat least one splint oligo comprises a functional nucleic acid, orwherein at least one splint oligo encodes information other thaninformation on a chemical library monomer. What is provided is the abovemethod, further comprising the step of coupling of at least one DNAbarcode module by way of click chemistry, wherein the step does not useany splint oligo.

Briefly stated, the present disclosure provides a system for screeningchemical compounds, comprising: (a) A picowell array plate comprising aplurality of picowells, wherein each picowell has a top aperture thatdefines an opening at the top of the picowell, a bottom that is definedby a floor, wherein the top aperture is separated from the floor, andwherein a wall resides in between the top aperture and the floor; (b) Atleast one bead disposed in at least one picowell, wherein the at leastone bead comprises a plurality of substantially identical bead-bound DNAbarcodes, and a plurality of substantially identical bead-boundcompounds, (c) Wherein the at least one bead comprises a DNA barcodethat takes the form of either a concatenated DNA barcode or anorthogonal DNA barcode, and wherein if the DNA barcode takes the form ofa concatenated DNA barcode the concatenated DNA barcode is made using amethod that: (i) Uses click chemistry, or (ii) Uses a repeating cycle ofsteps, wherein the steps in the repeating cycle comprise using a splintoligo for annealing to a partially made DNA barcode, wherein theannealed splint oligo is used as a template for extending the partiallymade DNA barcode using DNA polymerase, and wherein the splint oligocontains bases that are complementary to a DNA barcode module that is tobe polymerized to the partially made DNA barcode.

In another aspect, what is provided is the above system, wherein the DNAbarcode comprises: (a) One or more DNA barcode modules wherein each ofthe one or more DNA barcode modules encodes information on the identityof a chemical library monomer, and (b) Optionally one or more functionalnucleic acids, and (c) Optionally, one or more nucleic acids that encodeinformation that a type of information other than information on theidentity of a chemical library monomer.

Moreover, what is provides is the above system, further comprising aplurality of caps, each capable of fitting into the opening of adifferent picowell, and each capable of minimizing or preventingevaporation of fluid that is inside of the picowell, and each capable ofminimizing or preventing leakage of fluid that is inside of thepicowell.

Also embraced is the above system, further comprising a plurality ofspherical caps, wherein each is capable of fitting into the aperture ofa picowell wherein the aperture is circular, and each capable ofminimizing or preventing evaporation of fluid that is inside of thepicowell, and each capable of minimizing or preventing leakage of fluidthat is inside of the picowell.

Also contemplated is the above system, wherein if the at least one beadcomprises a DNA barcode that takes the form of a concatenated DNAbarcode, the concatenated DNA barcode comprises: (i) A sequencing primerbinding site, (ii) A first DNA barcode module, (iii) A first annealingsite that is capable of hybridizing with a first oligonucleotide splint,wherein the first oligonucleotide splint is capable of being used toguide the enzymatic synthesis of a second DNA barcode module, (iv) Asecond DNA barcode module, (v) A second annealing site that is capableof hybridizing with a second oligonucleotide splint, wherein the secondoligonucleotide splint is capable of being used to guide the synthesisof a third DNA barcode, (vi) A third DNA barcode module, (vii) A thirdannealing site that is capable of hybridizing with a thirdoligonucleotide splint, wherein the third oligonucleotide splint iscapable of being used to synthesize a fourth DNA barcode.

In methods embodiments, what is provided is a method for screening acompound library for compounds having desired properties, comprising:(a) providing a plurality of beads, wherein each bead comprises aplurality of oligonucleotides attached to the bead surface and aplurality of substantially related compounds attached to the beadsurface, and wherein the sequence of the oligonucleotides attached tothe beads encodes the synthesis history of the plurality ofsubstantially related compounds attached to the bead surface; (b)incorporating the plurality of beads in an assay for desired propertiesof compounds in the compound library; (c) capturing a signal from atleast one bead, wherein the signal reflects the performance of thecompounds on the bead in the assay; (d) sequencing the plurality ofoligonucleotides attached to the at least one bead for which assaysignal was also captured, without removing the oligonucleotides from thebead; and (e) identifying at least one compound from the sequencingreadout of step (d) and relating it to its corresponding assayperformance captured in the signal of step (c).

In further detail, what is embraced is the above method, wherein theassay comprises a binding assay, or wherein the assay comprises anactivity assay, or wherein the assay comprises a competitive bindingassay or a competitive inhibition assay, or wherein the assay comprisesinteraction of untethered compounds with other assay reagents, whereinthe untethered compounds are compounds released from the bead surface,or wherein the compounds are released by cleaving a cleavable linkerthat connects the compounds to the beads, or wherein the assay occurs ina plurality of confined volumes, wherein nominally one bead is dispersedper confined volume.

In another aspect, what is further contemplated, is the above method,wherein the confined volume comprises an aqueous droplet, or

wherein the aqueous droplet is suspended in an oil medium or ahydrophobic liquid medium, or wherein the confined volume comprises apicowell, or wherein the picowells are organized in a regular array, orwherein the plurality of confined volumes are organized in a regulararray.

Moreover, what is further embraced is the above method, wherein theconfined volume comprises a layer of adherent aqueous medium around thebead, wherein the bead is suspended in a hydrophobic medium, and theabove method, wherein the assay reagents are washed away beforesequencing the oligonucleotides. And the above method wherein thesequencing step (d) is performed before the assay step (b). What is alsoprovided is the above method, wherein the oligonucleotides on the beadsare removed after the sequencing step, but before the assay step.Moreover, further contemplated is the above method, wherein the removingof the oligonucleotide comprises an enzymatic digestion, a chemicalcleavage, a thermal degradation or a physical shearing, and the abovemethod, wherein the binding assay comprises binding of RNA molecules tothe beads, and the above method, wherein the signal from the beadcomprises sequencing of the bound RNA molecules.

In yet another aspect, what is provided is the above method, wherein thebinding assay comprises a fluorescently labeled binding assay, whereinthe molecules binding to the compounds on the beads comprisefluorophores, or the above method, wherein the binding assay comprisesnucleic-acid labeled binding assay, wherein the molecules binding to thecompounds on the beads comprise nucleic-acid tags, wherein further thesignal from the assay comprises sequencing of the nucleic acid tagsattached to the molecules binding to the compounds on the beads.

In yet a methods embodiment relating to properties, what is provided isthe above method, wherein the desired properties include one or more of:(i) Inhibiting or stimulating the catalytic activity of an enzyme, (ii)Stimulating Th1-type immune response, as measurable by cell-based assaysor by in vivo assays, (iii) Stimulating Th2-type immune response, asmeasurable by cell-based assays or by in vivo assays, (iv) InhibitingTh1-type immune response, as measurable by cell-based assays or by invivo assays, (v) Inhibiting Th2-type immune response, as measurable bycell-based assays or by in vivo assays, (vi) Stimulating or inhibitingubiquitin-mediated degradation of a protein, as measurable by purifiedproteins, by cell-based assay, or by in vivo assays.

In a system embodiment, what is provided is a system for screening acompound library for a compound having a desired activity, comprising:(a) a sample compartment for receiving a plurality of compound-attached,oligonucleotide-encoded beads; (b) a plurality of encapsulationcompartments within the sample compartment, each encapsulationcompartment nominally comprising a single bead dispersed in an assaymedium, wherein further the assay medium comprises reagents whoseinteraction with the compounds on the beads is being assayed resultingin a measurable signal; (c) a detector for measuring signals; (d) asequencing platform; and (e) a user interface for receiving one or morecommands from a user. Also provided is the above system, wherein theencapsulation compartment comprises a liquid droplet. In another aspect,provided is the above system, wherein the encapsulation compartmentcomprises a picowell, or wherein further the encapsulation compartmentcomprises assay reagents, or wherein the detector comprises an opticaldetector, or wherein the sequencer comprises the optical detector.

In one aspect, the disclosure features a method for perturbing a cellby: (a) providing a nucleic-acid encoded perturbation and confining acell with the nucleic-acid encoded perturbation; (b) contacting the cellwith the nucleic-acid encoded perturbation in a confined volume, whereinthe perturbation initiation and dose are controlled; (c) incubating thecell with the nucleic-acid encoded perturbation for a specified periodof time; and (d) transferring the nucleic acid that encodes thenucleic-acid encoded perturbation to the cell.

In some embodiments of this aspect, the nucleic-acid encodedperturbation is a nucleic acid encoded compound or drug molecule. Insome embodiments, the nucleic-acid encoded perturbation is a DNA-encodedlibrary.

In some embodiments, the perturbation and the nucleic acid encoding theperturbation are unattached and free in solution. In some embodiments,the perturbation and the nucleic acid encoding the perturbation areattached to each other. In some embodiments, the perturbation and thenucleic acid encoding the perturbation are attached to the samesubstrate but not to each other. In some embodiments, the attachment ofthe perturbation to the substrate and the attachment of the nucleic acidto the substrate are cleavable attachments. In particular embodiments,the cleavable attachment is selected from the group consisting of aphotocleavable attachment, a temperature cleavable attachment, a pHsensitive attachment, an acid cleavable attachment, a base cleavableattachment, a sound cleavable attachment, a salt cleavable attachment, aredox sensitive attachment, or a physically cleavable attachment.

In some embodiments of this aspect of the disclosure, confining the celland the perturbation comprises a droplet encapsulation, an emulsionencapsulation, a picowell encapsulation, a macrowell encapsulation, aphysical attachment, a bubble encapsulation, or a microfluidicconfinement.

In some embodiments, the control over the perturbation comprisescontrolling light exposure, controlling temperature exposure,controlling pH exposure, controlling time exposure, controlling soundexposure, controlling salt exposure, controlling chemical or physicalredox potential, or controlling mechanical-agitation exposure.

In particular embodiments, the incubation comprises exposing the cell tothe perturbation after cleaving the perturbation from the substrate orafter cleaving the nucleic acid from the substrate. In some embodiments,the incubation comprises exposing the cell to the perturbation withoutcleaving the perturbation from the substrate or without cleaving thenucleic acid from the perturbation.

In some embodiments, transferring the nucleic acid that encodes thenucleic-acid encoded perturbation to the cell comprises attaching thenucleic acid to the cell surface of the cell. In particular embodiments,attaching the nucleic acid to the cell surface of the cell comprisesintercalating the nucleic acid into the cell membrane. In particularembodiments, attaching the nucleic acid to the cell surface of the cellcomprises attaching the nucleic acid to a biomolecule on the cellsurface. In particular embodiments, the biomolecule is a protein or acarbohydrate. In other embodiments, attaching the nucleic acid to thecell surface of the cell comprises attaching through an optional tag onthe nucleic acid.

In another aspect, the disclosure features a method for perturbing acell with a perturbation and encoding the cell with the identity of theperturbation. The method includes: (a) providing a bead-bound DNAencoded library; (b) confining a cell with the bead-bound DNA encodedlibrary, wherein the bead-bound DNA encoded library comprises one ormore copies of a combinatorially synthesized compound and one or morecopies of an encoding nucleic acid tag, wherein the compound and theencoding nucleic acid are attached to a bead, wherein the encodingnucleic acid encodes the identity of the compound, and wherein thebead-bound DNA encoded library and the cell are confined in a confiningvolume; (c) releasing the compound from the bead and incubating thecompound with the cell inside the confining volume; (d) optionallyreleasing the encoding nucleic acid tag from the bead; and (e) attachingthe encoding nucleic acid tag to the cell, thereby preserving theidentity of the compound through the encoding nucleic acid tag attachedto the cell.

In yet another aspect, the disclosure features a method for perturbing acell, encoding the cell with the identity of the perturbation, andmeasuring a response of the cell to the perturbation. The methodincludes: (a) contacting a cell with a bead-bound DNA encoded library ina first confined volume, wherein the bead-bound DNA encoded librarycomprises one or more copies of a combinatorially synthesized compoundand one or more copies of an encoding nucleic acid tag, wherein thecompound and the encoding nucleic acid are attached to a bead, andwherein the encoding nucleic acid encodes the identity of the compound;(b) releasing the compounds in the library from the bead and incubatingthe compounds in the library with the cell inside the first confinedvolume; (c) optionally releasing the encoding nucleic acid tag from thebead inside the first confined volume; (d) capturing the encodingnucleic acid tag to the cell surface of the cell, whereby the cell isexposed to the compound in the library and the identity of the compoundexposed is captured on to the cell surface; (e) releasing the cell fromthe first confining volume, wherein the encoding nucleic acid tags areattached to the cell and the encoding nucleic acid tag encodes theidentity of the compound the cell is exposed to; (f) capturing apreviously perturbed and nucleic acid tagged cell with aresponse-detection bead in a second confined volume, wherein the cell isexposed to a lysis condition that exposes the cellular content of thecell to the response-capture bead, wherein the response-capture beadcomprises capture probes that capture the cellular content and thenucleic acid tag that encodes the perturbation in the previouslyperturbed and nucleic acid tagged cell; (g) incubating theresponse-capture bead with the lysed cell in the second confiningvolume, thereby capturing both cellular content and the nucleic acid tagthat encodes the perturbation on to the response-capture bead; (h)optionally converting the response of the cell to the perturbation to anucleic acid signal, wherein the response of the cell to theperturbation is not a nucleic acid signal; and (i) sequencing thenucleic acid tag attached to the response-capture bead, therebycorrelating the identity of the perturbation to the response of the cellto the perturbation.

In still another aspect, the disclosure features a method for perturbinga cell and capturing a response of the cell to the perturbation by: (a)providing an array of picowells and a library of functionalizedperturbation beads, wherein the picowells are capable of accommodating asingle cell and a single functionalized perturbation bead, wherein eachfunctionalized perturbation bead comprises a different plurality ofsubstantially identical releasable compounds and a plurality ofnucleotide barcodes that encodes the compounds, wherein the nucleotidebarcodes are functionalized barcodes capable of capturing cellularcontent of the cell, wherein the cellular content of cell comprisescellular response to the perturbations contained in the functionalizedperturbation beads; (b) capturing single cells into each picowell of thepicowell array; (c) capturing single functionalized perturbation beadsto the picowells containing single cells; (d) releasing the compoundsfrom the functionalized perturbation beads and incubating the cells withthe released compounds, wherein the compounds between picowells haveminimal diffusion; (e) lysing the cells to release the cellularcontents; (f) capturing one or more components of the cellular contentonto functionalized oligonucleotides on the functionalized perturbationbeads, wherein the capturing comprises hybridization and enzymaticextension to combine nucleotide barcodes with nucleic acid elements ofthe cellular content, thereby forming a hybrid of the nucleotide barcodeand the nucleic acid element of the cellular content; and (g) releasingthe hybrid, collecting the hybrid from the library of functionalizedperturbation beads, and sequencing the hybrid, thereby relating theperturbation to the cellular response to the perturbation.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1. Concatenated-style bead. In concatenated-style bead, the DNAbarcode takes the form of all of the DNA barcode modules connected toeach other in a single chain, together with any other nucleic acids thathave functions, such as primer annealing sites, as a spacer, orinformation on date of manufacture. The numbers on this figure are notstructure numbers. The numbers refer to the sequence of “DNA barcodemodules” in the DNA barcode.

FIG. 2. Orthogonal-style bead. In orthogonal-style bead, the DNA barcodetakes the form of all of the DNA barcode modules, where the DNA barcodemodules do not occur together in a single chain, but instead occurseparately linked to different positions on the bead. The numbers onthis figure are not structure numbers. The numbers refer to the sequenceof “DNA barcode modules” in the DNA barcode.

FIGS. 3A-3I. Cleavable linkers, conditions for cleavage (UV light orchemical), and cleavage products. Information from, Yinliang Yang (2014)Design of Cleavable Linkers and Applications in Chemical Proteomics.Technische Universitat Munchen Lehrstuhl fur Chemie der Biopolymere. Thealphabet letter to the left of each linker is from this reference.

FIG. 4. Exemplary amino acid derivatives for the compositions andmethods of the present disclosure.

FIGS. 5A-5H. The photograph discloses increases in degradation of afusion protein, inside HeLa cells, with increasing concentrations ofadded lenalidomide. Top: Expression of IKZF1/GFP fusion protein. Bottom:Expression of mScarlett® control. Lenalidomide was added at zero, 0.1,1.0, or 10 micromolar.

FIGS. 6A-6H. The photograph discloses increases in degradation of afusion protein, inside HeLa cells, with increasing concentrations ofadded lenalidomide. Top: Expression of IKZF3/GFP fusion protein. Bottom:Expression of mScarlett control. Lenalidomide was added at zero, 0.1,1.0, or 10 micromolar.

FIG. 7. Methods and reagents for creating bead-bound DNA barcode. Themost accurate description of “DNA barcode” is the sum of all of theinformation that is contained in the sum of all DNA barcode modules. Butfor convenience, the term “DNA barcode” is used herein to refer to thesum of all of the information of all of the DNA barcode modules plus anyadditional nucleic acids that provide information such as step number,or general type of chemical monomers that make up the bead-boundcompound, and plus any additional nucleic acids that serve a function,such as linker, sequencing primer binding site, hairpin with sequencingprimer binding site, or spacer. Where a DNA barcode is made, at least inpart, by way of click chemistry, the DNA barcode may include residualchemical groups from the click chemistry reactions.

FIG. 8. Structure of Alexa Fluor® 488. A goal of this figure is toidentify the compound without having to resort to using the trade name.

FIG. 9. Simplified diagram of bead-bound release-monitor. Therelease-monitor provides the user with a measure of the concentration ofthe soluble compound, following UV-induced release of the compound fromthe bead. In a preferred embodiment, one type of bead is dedicated tobeing a release-monitor, that is, this bead does not also containbead-bound compound and does not also contain bead-bound DNA library.“PCL” is photocleavable linker.

FIG. 10. Detailed diagram of bead release-monitor.

FIG. 11. Chemical synthesis of bead release-monitor.

FIG. 12. Amine-functionalized bead with bifunctional linker, where thelinker includes a lysine residue.

FIG. 13. Steps of chemical synthesis of lenalidomide modified with afirst type of carboxyl group.

FIG. 14. Steps of chemical synthesis of lenalidomide modified with asecond type of carboxyl group.

FIG. 15. Steps of chemical synthesis of lenalidomide modified with athird type of carboxyl group.

FIG. 16A, FIG. 16B, FIG. 16C. Lenalidomide analogues.

FIG. 17. Steps of chemical synthesis of a deoxycytidine analoguesuitable for click-chemistry synthesis of a DNA barcode.

FIGS. 18A, 18B, and 18C. Caps for placing over the top of picowells andfor sealing the picowells. FIG. 18A shows active cap, where compound isreleasable by way of cleavable linker. FIG. 18B shows another type ofactive cap, where a reagent such as an antibody is bound. The boundreagent can be permanently linked, it can be linked by a cleavablelinker, or it can be bound by way of hydrogen bonds and be releasablemerely by exposure to the solution in the picowell followed by diffusionaway from the active cap and into this solution. FIG. 18C shows apassive cap, which can be used to absorb, adsorb, collect, or capturemetabolites from the solution in the picowell. The absorbed metabolitescan subsequently be analyzed.

FIGS. 19A, 19B, 19C, and 19D. FIG. 19A Picowell plate without caps overthe picowells. FIG. 19B. Picowell plate with a cap over each picowell.FIG. 19C. Polyacrylamide solution being poured over the picowell platethat has one cap securely fastened over each picowell. Thepolyacrylamide then seeps into the porous cap, solidifies, and forms astable adhesion to each cap. FIG. 19D. The solidified polyacrylamide“roof” is then peeled off from the picowell plate, bringing with it eachcap. The metabolites transferred from the picowell solution and absorbedinto each cap can then be analyzed. Preferably, the solution that ispoured over the picowell plate and over the bead becomes a hydrogel, andpreferably the bead is made from a hydrogel.

In exclusionary embodiments, the present disclosure can exclude asystem, microtiter plate, microtiter plate with microwells, nanowells,or picowells, and related methods, where at least one well is capped,and where a liquid polymer solution is poured over the plate and overthe capped wells. Also, what can be excluded is the above where theliquid polymer has polymerized to form a solid polymer that adheres toeach cap. Also, what can be excluded is the method and resultingcompositions, where the solid polymer is torn away, removing with it theadhering caps.

FIG. 20. Map of circular plasmid used for integrating IKZF1 gene intogenome of a cell. The plasmid is: IKZF1 mNEON-p2a-mScarlet-w3-2FB (9081base pairs). IKZF1 encodes the Ikarus protein.

FIG. 21. Map of circular plasmid used for integrating IKZF3 gene intogenome of a cell. The plasmid is: IKZF3 mNeon-p2a-mScarlet-w3-2FB (9051bp). IKZF3 encodes the Aiolos protein.

FIG. 22. Chemical monomers (compounds 1-6) and their DNA barcodes.

FIG. 23. Chemical monomers (compounds 7-10) and their DNA barcodes.

FIG. 24. Chemical monomers (compounds 11-16) and their DNA barcodes.

FIG. 25. Chemical monomers (compounds 17-21) and their DNA barcodes.

FIG. 26. Chemical monomers (compounds 22-16) and their DNA barcodes.

FIG. 27 Chemical monomers (compounds 27-30) and their DNA barcodes.

FIG. 28. Sequencing a bead-bound DNA barcode. The figure disclosesintensity of fluorescent signal for each of five consecutive bases,where the five consecutive bases are part of a bead-bound DNA barcode.

FIG. 29. Stepped picowell.

FIGS. 30A-30F. Time course of release of the fluorophore from the bead.This shows operation of the bead-bound release monitor, acquisition offluorescent data at t=0 seconds, t=1 seconds, t=11 seconds, and t=71seconds.

FIGS. 31A-31B. Emission data resulting after catalytic action ofaspartyl protease on quencher-fluorophore substrate.

FIG. 32. Drawings of cross-section of picowells, illustrating varioussteps.

FIGS. 33A-33F. Titration data showing how increase in UV dose results ingreater cleavage of fluorophore from the bead. In layperon's terms, thisshows how a more powerful swing of the axe influences chopping thefluorophore from the bead (the power of the UV does is measured inJoules per centimeter squared). The notation “Exposure” refers only to aparameter when taking the photograph. It is just exposure time, whentaking the photograph (it does not refer to exposure time of the lightdoing the cleaving, or to the light doing the exciting).

FIG. 34. TAMRA concentration versus luminous flux. What is shown isconcentration of free TAMRA, following release after exposure to UVlight at 365 nm.

FIG. 35 provides a hand-drawings of the quencher-fluorophore substrate,and of cleavage of this substrate by the enzyme, with consequentinhibition of enzyme. Also shown is the molecular structure ofbead-bound pepstatin-A, and bead-bound Fmoc-valine (negative control).

FIG. 36. Steps in preparing beads for use in eventual capture of mRNAfrom lysed cells, with subsequent manufacture of cDNA library. Thisfigure also occurs in one of the Provisional applications (Compositionsand Method for Screening Compound Libraries on Single Cells), from whichpriority of the present application is claimed.

FIG. 37. Tagging cells with DNA barcode, where tagging is by way of alipid that embeds in the cell membrane. This figure also occurs in oneof the Provisional applications (Compositions and Method for ScreeningCompound Libraries on Single Cells), from which priority of the instantapplication is claimed.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the” include their corresponding pluralreferences unless the context clearly dictates otherwise. All referencescited herein are incorporated by reference to the same extent as if eachindividual patent, and published patent application, as well as figures,drawings, sequence listings, compact discs, and the like, wasspecifically and individually indicated to be incorporated by reference.

ABBREVIATIONS

Table 1 provides abbreviations and non-limiting definitions.

TABLE 1 Abbreviations and non-limiting definitions ACN AcetonitrileAMPSO 3-[(1,1-dimethyl-2-hydroxyethyl) amino]-2-hydroxypropanesulphonicacid. AMPSO is one of the “Good buffers” ((1966). Hydrogen Ion Buffersfor Biological Research. Biochemistry. 5: 467-477). Aperture As usedherein, the term aperture is used herein to refer to a physicalsubstance that defines an opening and, more specifically, to the minimalamount of physical substance that is capable of defining an opening.Without implying any limitation, this minimal amount of physicalsubstance preferably takes the form of a ring-shaped section of a wall.Without limitation, the aperture can be considered to be a ring-shapedsection of a wall, where the thickness of the section is about 0.2 nm,about 0.5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about200 nm, about 500 nm, about 1 micrometer (um), about 2 um, about 5 um,and so on, where this thickness measurement is in the radial directionextending away from an axis, and where the axis is defined by theopening. 1-AP 1-Azidopyrene ATB Active tuberculosis Barcode The term“DNA barcode” can refer to a polynucleotide that identifies a chemicalcompound in its entirety while, in contrast, “DNA barcode module” canrefer to only one of the monomers that make up the chemical compound. Ashort definition of a “DNA barcode module” is that it identifies achemical library monomer. However, a “DNA barcode module” can be used toidentify the history of making that particular monomer. A longerdefinition of a “DNA barcode module” is as follows. Each of thefollowing chemical library monomers need to be identified by a different“DNA barcode module.” Even the first reaction and the second reactionhave the same reactants (A and B), a different DNA barcode module isused, because the products are different (the products being either C orD). Also, even though the first reaction and the third reaction resultin the same product (the product being “C”), a different DNA barcodemodule is used, because the reactants are different (the reactants beingeither A + B, or X + Y). Reaction Condition A + B → C Reaction conditionA, for example, with methane solvent A + B → D Reaction condition A, forexample, with methylene chloride solvent X + Y → C BiNAP BiNAP takes theform of two naphthalene groups attached to each other by way of acarbon-carbon bond between the 1-carbon of the first naphthalene and the1-carbon of the second naphthalene. Each naphthalene group also containsan attached PPh₂ group, where the PPh₂ group is attached to thenaphthalene's 2-carbon. PPh₂ takes the form of a phosophate group, towhich is attached two phenyl groups. In BiNAP, the phosphate is situatedin between the naphthalene and the PPh₂. BTPBB Bis-Tris propane breakingbuffer BTPLB Bis-Tris propane ligation buffer BTPWB Bis-Tris propanewash buffer Cap A cap is an object that can serve as a plug, a stopper,a seal, and the like, for placing in stable contact with a microwell,nanowell, or picowell. The cap can be spherical, ovoid, cubical, cubicalwith rounded edges, pyramidal, pyramidal with rounded edges, and so on.Unless specified otherwise, the stated shape is the shape prior topartial insertion or prior to full insertion into the picowell.Preferably, when in use the cap is partially inserted into the picowellto form a seal. In some embodiments, the cap may be loosely set on topof the picowell without any partial insertion. Compound The term“compound” is used here, without implying any limitation, to refer to acompleted chemical that is synthesized by connecting a plurality ofchemical monomers to each other, by way of solid phase synthesis on abead. Generally, the term “compound” refers to the completed chemicalthat is to be tested for activity by way of an assay. The term“compound” is not intended to include any linkers that mediate bindingof the completed chemical to the bead, and is not intended to includeany protecting groups that are to be cleaved off, though it isunderstood that a “compound” that has a protecting group may havepharmaceutical activity. The term “compound” is NOT used to refer tobead-bound chemicals where not all of the chemical monomers have beenconnected. If the term “compound” is used in some other context herein,the skilled artisan will be able to determine if this description isrelevant or not. COMU 1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (CAS no.1075198-30-9) Concatenated A DNA barcode that is “concatenated,” takesthe form where all of the DNA barcode nucleic acid modules are part ofthe same polymer. When a bead contains a DNA barcode taking the barcodeconcatenated form, all of the information from all of the constituentDNA barcode modules are present on the polymer that is attached to asingle attachment site on the bead. Concatenated DNA refers to“end-to-end ligation” or “end-to-end joining” (Farzaneh (1988) NucleicAcids Res. 16: 11319-11326; Boyer (1999) Virology. 263: 307-312). Incontrast, the word “catenated” refers to two circles of DNA that arelinked to each other as in a chain (Baird (1999) Proc. Nat'l. Acad. Sci.96: 13685-13690). CuAAC Copper-catalyzed azide-alkyne cycloaddition CRBClick reaction buffer DAF Diazofluorene DBCO Dibenzocyclooctyne DBU1,8-diazabicyclo [5.4.0] undec-7-ene DCE 1,2-Dichloroethane DCMDichloromethane DESPS DNA encoded solid-phase synthesis DIC Diisopropylcarbodiimide DIEA N,N′-diisopropylethylamine DMA Dimethylacetamide DMAP4-Dimethylaminopyridine DMF Dimethylformamide DI Deionized DTTDithiothreitol EDC Ethyl-dimethylaminopropyl-carbodiimide ELISA Enzymelinked immunosorbent assay FMOC 9-Fluorenylmethoxycarbonyl FMOC-PCL-4-[4-[1-(9-Fluorenylmethyloxycarbonylamino)ethyl]-2-methyoxy-5-nitrophenoxy]OH butanoic acid (CAS No. 162827-98-7) Functional In the context of abead-bound DNA barcode, and in the context of manufacturing a nucleicacid bead-bound DNA barcode, the term “functional nucleic acid” refersto nucleic acids with an active biochemical function (a function thattakes advantage of hydrogen bonds, of hydrophobic interactions, ofhydrophilic interactions, of interactions with enzymes, etc.). Thefunction can be a spacer that establishes a distance between ahydrophobic bead and a primer binding site. The primer binding sitepreferably occurs in a hydrophilic environment for supporting activityof DNA polymerase. Also, the function can be a primer binding site, ahairpin bend, or the annealing site for a “splint oligo.” This is incontrast to “informational nucleic acids,” which store information(which “encode”) information on the identity of a corresponding chemicalmonomer. HDNA Headpiece DNA HTS High throughput screening INA5-Iodonaphthalene-1-azide LC Liquid chromatography LTB Latenttuberculosis MDM2 Murine Double Minute 2 Mtt 4-Methyltrityl NCL hits;NCL NCL refers to a mixture of sera from latent tuberculosis patients(this accounts for the pool letter “L”) and sera from negative control,healthy human subjects (this accounts for the letter “NC”) NHSN-Hydroxysuccinimide. NHS chemistry can be used to attach tetrazine tofree amino groups of, for example, antibodies (van Buggenum, Gerlach,Mulder (2016) Scientific Reports. 6: 22675. Nucleic acid The term“nucleic acid” can refer to a single nucleic acid molecule, or tomodified nucleic acids, such as a nucleic acid bearing a fluorescenttag. Also, the term “nucleic acid” can be used to refer individualcontiguous stretches of nucleotides within a longer polynucleotide.Here, the term “nucleic acid” makes it more convient to refer to theseindividual stretches within a longer polynucleotide, for example, aswhen the polynucleotide comprises a first nucleic acid that is aprimer-binding site, a second nucleic acid that is a DNA barcode module,and a third nucleic acid that identifies the step number in a multi-steppathway of synthesis. OP Oligo pair. Oligo pair can refer to a reagentthat takes the form of a slipped heteroduplex, for example, an aqueoussolution of a slipped heteroduplex. Orthogonal A DNA barcode that is“orthogonal,” takes a form where each of the DNA barcode nucleic acidmodules occupies a different attachment site on the bead. When a beadcontains a DNA barcode barcode taking the orthogonal form, theacquisition of all of the information of a compound's DNA barcoderequires separately sequencing each of the attached DNA barcode modules.In other words, with an “orthogonal” nucleic acid barcode, each andevery one of the DNA barcode modules that makes up the DNA barcode isdispersed over different attachment sites on the same bead. OSu (OSu isN-Hydroxysuccinimide the same as NHS) OXYMA Ethyl2-cyano-2-(hydroxyamino)acetate Parallel The term “parallel” refers tothe situation where chemical monomers are covalently attached to a bead,one by one, to create a bead-bound compound, and where nucleic acidbarcode modules are also covalently attached to the same bead, one byone, to create a bead-bound nucleic acid barcode. The chemical reactionthat attaches each chemical monomer is not carried out at exactly thesame time as the reaction (chemical or enzymatic) that attaches eachnucleic acid barcode module. Instead, these two reactions are staggered,so that the parallel synthesis involves first attaching the chemicalmonomer, and then attaching the corresponding nucleic acid barcodemodule. Alternately, the staggered reaction can involve first attachingthe nucleic acid and then attaching the corresponding chemical monomer.What is corresponding in this situation, is that each nucleic acidbarcode module serves to identify the chemical monomer that is attachedin the same round of parallel synthesis. PCL Photocleavable linker PEGPolyethylene glycol PDMS Polydimethylsiloxane qPCR Quantitativepolymerase chain reaction Picowell Without implying any limitation onthe presend disclosure, the term “picowell” can be used to refer to awell or cavity in a plate that contains an array of picowells, forexample, over 50,000 picowells, over 100,000 picowells, over 200,000picowells, over 500,000 picowells, and so on. Typically, the volume of apicowell (not including the volume of any beads that might be in thepicowell), is about 0.2 picoliters (pL), about 0.5 pL, about 1.0 pL,about 2.0 pL, about 5.0 pL, about 10 pL, about 20 pL, about 30 pL, about40 pL, about 50 pL, about 75 pL, about 100 pL, about 200 pL, about 300pL, about 400 pL, about 500 pL, about 600 pL, about 700 pL, about 800pL, about 1000 pL, about 10,000 pL, about 100,000 pL, about 1,000,000pL, or in a volume range defined by any of the above two values, forexample, about 0.5 to 2.0 pL. The volumes for any “nanowell” and“microwell” can be set as above (except with the term “pico” replaced bynano or micro). Unless specified otherwise, explicitly or by context,the present disclosure refers to picowells (rather than to nanowells ormicrowells). RAM Rink Amide RCA Rolling circle amplification RT Roomtemperature SPS Solid phase synthesis Slipped Slipped heteroduplexstructure takes the form of a first strand of ssDNA and a secondheteroduplex strand of ssDNA, where a dozen nucleotides at the 5′-end ofthe first strand of ssDNA structure are complementary to a dozennucleotides at the 5′-end of the second strand of ssDNA, and where thefirst strand of ssDNA is binds to the second strand of ssDNA by way of adozen complementary base pairings that involve the respective5′-termini. The number “dozen” is purely exemplary and is not limiting.Alternatively, the slipped heteroduplex structure could be maintained asa hybridized duplex, by way of complementary base pairing at the 3′-endof the first strand of ssDNA and the 3′-end of the second strand ofssDNA. The term “slipped heteroduplex structure” can alternatively becalled a “staggered heteroduplex structure.” The term “slipped” does notimply that the heteroduplex is slippery (can shift position0 as might bethe case with a duplex formed when oligo[C] hybridizes to oligo[G], orwhen oligo[A] hybridizes to oligo[T]. TB Tuberculosis TBE Tris borateEDTA TBAI Tetrabutyl ammonium iodide. TBTATris[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine TCEPTris(2-carboxyethyl)phosphine. Reducing agent that can cleave disulfidebonds. TCO Trans-cyclooctene TEAA Triethylammonium acetate TEV proteaseTobacco Etch Virus protease TFA Trifluoroacetic acid TID3-(trifluoromethyl)-3-(m-iodophenyl) diazirine TIPS Triisopropyl silaneTM Temperature of melting TMP 2,4,6-Trimethylpyridine QSY7 Xanthylium,9-[2-[[4-[[2,5-dioxo-1-pyrrolidinyl)oxy] carbonyl]-1-piperidinyl]sulfonyl]phenyl]-3,6-bis(methylphenylamino)-, chloride (CAS No.304014-12-8) TAMRA 5(6)Carboxytetramethyl rhodamine

Reagents, kits, enzymes, buffers, living cells, instrumentation, and thelike, can be acquired. See, for example, Sigma-Aldrich, St. Louis, Mo.;Oakwood Chemical, Estill, S.C.; Epicentre, Madison, Wis.; Invitrogen,Carlsbad, Calif.; ProMega, Madison, Wis.; Life Technologies, Carlsbad,Calif.; ThermoFisher Scientific, South San Francisco, Calif.; NewEngland BioLabs, Ipswich, Mass.; American Type Culture Collection(ATCC), Manassas, Va.; Becton Dickinson, Franklin Lakes, N.J.; Illumina,San Diego, Calif.; 10× Genomics, Pleasanton, Calif.

Barcoded gel beads, non-barcoded gel beads, and microfluidic chips, areavailable from 1CellBio, Cambridge, Mass. Guidance and instrumentationfor flow cytometry is available (see, e.g., FACSCalibur®, BDBiosciences, San Jose, Calif., BD FACSAria II® User's Guide, part no.643245, Rev. A, December 2007, 344 pages).

A composition that is “labeled” is detectable, either directly orindirectly, by spectroscopic, photochemical, fluorometric, biochemical,immunochemical, isotopic, or chemical methods, as well as with methodsinvolving plasmonic nanoparticles. For example, useful labels include,³²P, ³³P, ³⁵S, ¹⁴C, ³H, ¹²⁵I, stable isotopes, epitope tags, fluorescentdyes, Raman tags, electron-dense reagents, substrates, or enzymes, e.g.,as used in enzyme-linked immunoassays, or fluorettes (Rozinov and Nolan(1998) Chem. Biol. 5:713-728).

TABLE OF CONTENTS FOR DETAILED DESCRIPTION (I) Beads (II) One bead onecompound (OBOC) (III) Coupling nucleic acids to beads (IV) DNA barcodes(V) Coupling chemical compounds to beads (VI) Coupling chemical monomersto each other to make a compound (VII) Split and pool synthesis andparallel synthesis (VIII) Fabricating picowells (IX) Deposit beads intopicowells (X) Sequencing bead-bound nucleic acids in picowells (XI)Releasing bead-bound compounds from the bead (XII Biochemical assays forcompounds (XIII) Cell-based assays for compounds (I) BEADS

The methods and compositions of the present disclosure use beads, suchas monosized TentaGel® M NH₂ beads (10, 20, 30, etc., micrometers indiameter)-, standard TentaGel® amino resins (90, 130, etc. micrometersin diameter), TentaGel Macrobeads® (280-320 micrometers in diameter)(all of the above from Rapp Polymere, 72072 Tubingen, Germany). Thesehave a polystyrene core derivatized with polyethylene glycol (Paulick etal (2006) J. Comb. Chem. 8:417-426). TentaGel® resins are graftedcopolymers consisting of a low crosslinked polystyrene matrix on whichpolyethylene glycol (PEG) is grafted. Thus, the present disclosureprovides beads or resins that are modified to include one or both of aDNA barcode and a compound, where the unmodified beads take the form ofgrafted copolymers consisting of a low crosslinked polystyrene matrix onwhich polyethylene glycol (PEG) is grafted.

TentaGel® is characterized as, “PEG chains of molecular masses up to 20kilo Dalton have been immobilized on functionalized crosslinkedpolystyrenes. Graft copolymers with PEG chains of about 2000-3000 Daltonproved to be optimal in respect of kinetic rates, mobility, swelling andresin capacity.” (Rapp Polymere, Germany). Thus, the present disclosureprovides beads or resins that take the form of graft copolymers with PEGchains of about 2000-3000 Daltons. Regarding swelling, Comellas et alprovides guidance for measuring the ability of a bead to swell, forexample, when soaked in DCM, DMF, methyl alcohol, water, or a bufferused in enzyme assays (Comellas et al (2009) PLoS ONE. 4:e6222 (12pages)). The unit of swelling is milliliters per gram of bead.

In an alternate bead embodiment, the present disclosure uses a resinwith a PEG spacer is attached to the polystyrene backbone via an alkyllinkage, and where the resin is microspherical and monosized (TentaGel®M resin).

In yet an alternate bead embodiment, the present disclosure uses a resinwith a PEG spacer attached to the polystyrene backbone via an alkyllinkage, where the resin type exists in two bifunctional species: First,surface modified resins: the reactive sites on the outer surface of thebeads are protected orthogonally to the reactive sites in the internalvolume of the beads, and second, hybrid resins: cleavable andnoncleavable ligands are present in this support—developed forsequential cleavage (TentaGel® B resin).

Moreover, in another embodiment, the present disclosure uses a resinwhere a PEG spacer is attached to the polystyrene backbone via an alkyllinkage, and where the macrobead resin shows very large particlediameters and high capacities (TentaGel® MB resin). Also, the presentdisclosure uses a resin where the PEG spacer is attached to thepolystyrene backbone via a benzyl ether linkage. This resin can be usedfor immunization procedures or for synthesizing PEG modified derivatives(PEG Attached PEG-modified compounds) (TentaGEl® PAP resin).

Moreover, the beads can be, HypoGel® 200 resins. These resins arecomposites of oligoethylene glycol (MW 200) grafted onto a lowcross-linked polystyrene matrix (Fluka Chemie GmbH, CH-9471 Buchs,Switzerland).

In some embodiments amino functionalized polystyrene beads, without PEGlinkers, may be used, for instance, monosized polystyrene M NH₂microbeads (5, 10, 20 etc., micrometers in diameter, also from RappPolymere, 72072 Tübingen, Germany).

In some embodiments, compounds may be encapsulated within pores orchambers or tunnels within the beads, without covalent attachment to thebeads. Compounds may be diffused into or forced within such pores of thebeads by various means. In some embodiments the compounds may be loadedwithin the beads by diffusion. In some embodiments, high temperature maybe used to swell the beads and load compounds within the beads. In someembodiments, high pressure may be used to force compounds into thebeads. In some embodiments, solvents that swell the beads may be used toload compound within the beads. In some embodiments, vacuum or lowpressure may be used to partition compounds into beads. In someembodiments mild, or vigorous physical agitation may be used to loadcompounds into beads.

In such embodiments where the compounds are loaded onto beads withoutcovalent attachment, compounds may be unloaded from the bead by way ofdiffusion. In some embodiments, in a non-limiting fashion, temperature,pressure, solvents, pH, salts, buffer or detergent or combinations ofsuch conditions may be used to unload compounds out of such beads. Insome embodiments the physical integrity of the beads, for instance byuncrosslinking polymerized beads, may be used to release compoundscontained within such beads.

In exclusionary embodiments, the present disclosure can exclude anybead, and bead-compound complex, or any method, that involves one of theabove beads.

Beads of the present disclosure also include the following. Merrifieldresin (chloromethylpolystyrene); PAM resin (4-hydroxymethylphenylacetamido methyl polystyrene); MBHA resin (4-methylbenzhydrylamine);Brominated Wang resin (alpha-bromopriopiophenone); 4-Nitrobenzophenoneoxime (Kaiser) resin; Wang resin (4hydroxymethyl phenoxymethylpolystyrene; PHB resin (p-hydroxybenzyl alcohol; HMPA resin(4-hydroxymethyl phenoxyacetic acid); HMPB resin(4-hydroxymethyl-3-methoxy phenoxyl butanoic acid); 2-Chlorotritylresin; 4-Carboxytrityl resin; Rink acid resin (4-[(2,4-dimethoxypehenyl)hydroxymethyl) phenoxymethyl); Rink amide (RAM) resin “Knorr” resin(4-((2,4-dimethylphenyl) (Fmox-amino)methyl) phenoxyalkyl); PAL resin(5-[4-(Fmoc-amino) methyl-3,5-dimethoxyphenoxy] valeramidomethylpolystyrene); Sieber amide resin (9-Fmox-amino-xanthan-3-yl-oxymethyl);HMBA resin (hydroxymethyl benzoic acid); 4-Sulfamoylbenzoyl resin“Kenner's safety catch” resin (N-(4-sulfamoylbenzoyl)aminomethyhl-polystyrene); FMP-resin(4-(4-formyl-3-methoxyphenoxy)-ethyl) (see, ChemFiles Resins forSolid-Phase Peptide Synthesis Vol. 3 (32 pages) (Fluka Chemie GmbH,CH-9471 Buchs, Switzerland).

Beads of the present disclosure further include the above beads used aspassive encapsulants of compounds (passively hold compounds withoutcovalent linkage to the compound), and further comprising the following:unfunctionalized polystyrene beads; silica beads; alumina beads; porousglass beads; polyacrylamide beads; titanium oxide beads; alginate beads;ceramic beads; PMMA (polymethylmethacrylate) beads; melamine beads;zeolite beds; polylactide beads; deblock-copolymer micelles; dextranbeads, and others. Many of the beads listed in this paragraph may bepurchased from vendors such as Microspheres-Nanospheres, Cold Spring,N.Y. 10516, USA.

In addition to beads, vesicles or droplets may also be used as vehiclesfor delivering compounds for some embodiments of the present disclosure.Lipids, deblock-copolymers, tri-block copolymers or other membraneforming materials may be used to form an internal volume into whichcompounds may be loaded. Compounds may be released from theseencapsulated volumes by addition of detergent, mechanical agitation,temperature, salt, pH or other means. Water-in-oil droplet emulsions oroil-in-water droplet emulsions are yet other means to passivelyencapsulate compounds that may be delivered to assay volumes.

In all embodiments where passive encapsulation is used to delivercompounds, DNA tags may also be loaded passively, or alternatively, theDNA tags may be covalently attached to the beads, vesicles or droplets.

In exclusionary embodiments, the present disclosure can exclude anybeads or resins that are made of any one the above chemicals, or thatare made of derivatives of one any one of the above chemicals.

In embodiments, the beads can be spheroid and have a diameter of about0.1-1 micrometers, about 1-5 micrometers, about 1-10, about 5-10, about5-20, about 5-30, about 10-20, about 10-30, about 10-40, about 10-50,about 20-30, about 20-40, about 20-50, about 20-60, about 50-100, about50-200, about 50-300, about 50-400, about 100-200, about 100-400, about100-600, about 100-800, about 200-400, about 200-600, about 200-800micrometers, and so on.

Non-spheroid beads that are definable in terms of the above values andranges are also provided. For example, one of the axes, or one of theprimary dimensions (for example, a side) or one of the secondarydimensions (for example, a diagonal) may comprise values in the aboveranges. In exclusionary embodiments, the present disclosure can excludeany reagent, composition, system, or method, that encompasses spheroidbeads (or non-spheroid beads) falling into one or more of the abovevalues or ranges.

Chains of Beads.

In one embodiment, what is provided is a plurality of bead dimers, wherethe bead-dimer takes the form of two beads that are attached to eachother, and where one bead contains a plurality of attached nucleic acidbarcodes (either orthogonal nucleic acid modules, or concatenatednucleic acid modules), and the other bead contains a plurality ofattached compounds, where all of the compounds are substantially relatedto each other (or where all of the compounds are substantially identicalin chemical structure to each other). The bead dimer may be synthesizedby preparing the first bead that has the attached compounds, separatelypreparing the second bead that has attached nucleic acid barcodes, andthen linking the two beads together. In one aspect, the beads areattached to each other by a reversible linker, and in another aspect,the beads are attached to each other by a non-reversible linker.

Bead Permeability.

In embodiments, the present disclosure provides beads with variousranges or degrees of permeability. Permeability can be measured as thepercentage of the volume of the bead that is accessible by a solvent,where the unit of measurement is percentage of the bead's surface thattakes the form or pores, or where the unit of measurement is percentageof the bead's interior that takes the form of channels, networks, orchambers that are in fluid communication with the surface (and exteriormedium) of the bead. The present disclosure can encompass porous beadsor, alternatively, can exclude porous beads.

U.S. Pat. No. 9,062,304 of Rothberg discloses a bead with an exteriorand with interior regions. What is shown is “internal surfaces (poresurfaces),” and that “suitable pores will . . . exclude largermolecules,” and the option of “exploiting differential functionalizationof interior and exterior surfaces,” and various pore diameters, andpolymers such as poly(styrene sulfonic acid) and polystyrene. FIG. 1 ofRothberg provides pictures of surface of bead and pores of bead. U.S.Pat. No. 9,745,438 of Bedre provides transmission electron microscopeimage of porous bead. U.S. Pat. No. 5,888,930 of Smith provides scanningelectron micrograph of cross-section of porous bead. What is shown issphereical bead with small pores on surface and large pores inside,where bead is made from, e.g., polystyrene, polyacrylonitrile,polycarbonate, cellulose, or polyurethane. U.S. Pat. No. 5,047,437 ofCooke discloses sphereical poly(acrylonitrile) copolymer pore morphologywith skinless surface (FIG. 1) and bead that has exterior skin onsurface (FIG. 5). U.S. Pat. No. 4,090,022 of Tsao discloses porousopenings and internal void spaces, of cellulose beads.

Each of the above-identified patents, including all of the figures, isincorporated herein in its entirety, as though each was individuallyincorporated by reference in its entirety.

Without implying any limitation, exterior surface of a bead ormicroparticle can be determined by tightly wrapping the entire bead ormicroparticle with an elastic film. The bead or microparticle can bewrapped by way of a thought-experiment, or the wrapped bead can bedepicted by a drawing or photograph, or the bead can be wrapped inreality. Without implying any limitation, the exterior surface of thebead is that part of the bead that physically contacts the wrapping.

For example, the present disclosure provides a bead with poresaccounting for at least 1%, at least 2%, at least 5%, at least 10%, atleast 15%, at least 20%, at least 30%, at least 40%, of the surfacearea. Also, the present disclosure provides a bead where the volume ofthe internal channels or networks accounts for at least 1%, at least 2%,at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, ofthe total volume of the bead, and where the internal channels ornetworks are in fluid communication with the outside surface (andexterior medium) of the bead.

Moreover, the present disclosure provides a bead with pores accountingfor less than 1%, less than 2%, less than 5%, less than 10%, less than15%, less than 20%, less than 30%, less than 40%, of the surface area.Also, the present disclosure provides a bead where the volume of theinternal channels or networks accounts for less than 1%, less than 2%,less than 5%, less than 10%, less than 15%, less than 20%, less than30%, less than 40%, less than 50%, less than 60%, less than 70%, lessthan 80%, of the total volume of the bead, and where the internalchannels or networks are in fluid communication with the outside surface(and exterior medium) of the bead.

Iron-Core Beads.

The present disclosure encompasses iron-core beads or magnetic beads.These beads can be manipulated with magnets to move them from onereaction vessel to another reaction vessel, or from one container toanother container. Manipulations by robotics can be enhanced by usingthese beads. Methods of manufacture and use of magnetic beads areavailable (Szymonifka and Chapman (1994) Tetrahedron Letters.36:1597-1600; Liu, Qian, Xiao (2011) ACS Comb. Sci. 13:537-546; Alam,Maeda, Sasaki (2000) Bioorg. Med. Chem. 8:465-473).

In exclusionary embodiments, the present disclosure can exclude anybead, or any population of beads, where the bead or population meets oneof the above values or ranges.

Compound Loading into Beads

In many experiments it is advantageous to load pre-synthesized compoundsinto beads, where the beads may be used as vehicles for delivering thecompounds to an assay. Many of the standard techniques used for drugdelivery to biological specimens may be adapted to deliver compounds toassays (see, Wilczewska et al (2012) Nanoparticles as drug deliverysystems. Pharmacological Reports. 64:1020-1037, Kohane DS (2007)Microparticles and nanoparticles for drug delivery. Biotechnol. Bioeng.96: 203-209, Singh et al (2010) Microencapsulation: A promisingtechnique for controlled drug delivery. Res Pharm Sci. 5: 65-77).

In such embodiments where pre-synthesized compounds are loaded intobeads, the compounds may be held in traditional 96, 385 or 1536 wellmicrotiter plates. To these plates, beads may be added, into which thecompounds get loaded by diffusion or by other active loading methods. Inpreferred embodiments, the beads chosen for impregnation have pore sizesor percolation geometries that prevent immediate emptying of thecompounds when removed from the mother solution. The diffusion out ofthe beads may be enhanced by heat, pressure, additives or otherstimulants, if needed. In some embodiments, the compound-laden beads maybe capped in a manner that prevents leakage of the internal contentsuntil triggered by an external impulse. One method for capping theexteriors of porous beads involves adding lipids or amphiphilicmolecules to the bead-compound solution, such that the cavities exposedto the surface of the beads get sealed by a bilayer formed by theamphiphilic molecules. In some embodiments preformed vesicles may bemixed with the drug laden beads, such that upon agitating, the vesiclesrupture and the membranes reform over the surface of the drug-ladenbeads, thereby sealing them. Methods to perform such bead sealing aredescribed (see, Tanuj Sapra et al (2012) Nature Scientific Reportsvolume 2, Article No.: 848). Further experimental protocols to sealsilica beads are available in the report release by Sandia Laboratoriesby Ryan Davis et al, Nanoporous Microbead Supported Bilayers: Stability,Physical Characterization, and Incorporation of Functional TransmembraneProteins, SAND2007-1560, and the method bSUM, described by Hui Zheng etal (bSUM: A bead-supported unilamellar membrane system facilitatingunidirectional insertion of membrane proteins into giant vesicles) in J.Gen. Physiol. (2016) 147: 77-93.

In some embodiments utilizing pre-synthesized compounds, beads aregenerated from the compounds by addition of appropriate reagents, forinstance by adding lipids or di-block copolymers followed by agitation,whereby vesicles are formed containing the compounds in their interioror within the bilayer membrane. In some embodiments, the compounds maybe pushed through a microfluidic T junction to create aqueous phasedroplets in an oil phase, where the compounds are contained within theaqueous phase or at the interface between the aqueous phase and the oilphase. In some embodiments, the droplets formed may further bepolymerized, creating hydrogels, that are more rugged and stable tohandling than unpolymerized aqueous phase droplets. Droplet-basedencapsulation and assays are disclosed by, Oliver et al (2013) DropletBased Microfluidics, SLAS Discovery Volume: 19 issue: 4, page(s):483-496. Sol-gel encapsulation process may also be employed toencapsulate compounds within beads. Formation of sol-gel beads isdescribed in, Sol-gel Encapsulation of Biomolecules and Cells forMedicinal Applications, Xiaolin Wang et al (2015) Current Topics inMedicinal Chemistry. 15: 223.

One Bead One Compound (OBOC)

Methods used to manufacture combinatorial libraries involve three steps,(1) Preparing the library; (2) Screening the compounds in the library,and (3) Determining the structure of the compounds, for example, of allof the compounds or only of the compounds that provided an interestingresult with screening (see, Lam et al (1997) The One-Bead-One-CompoundCombinatorial Library Method. Chem. Rev. 97:411-448). An advantage ofsynthesizing compounds by way of a bead-bound synthesis, is that thecompound can be made rapidly by the “split-and-pool” method.

OBOC Combined with an Encoding Strategy.

Another feature of OBOC is that each bead can include, not only acompound but also an encoding strategy. Where bead-bound nucleic acidsare used for encoding a compound that is bound to the same bead, theterm “encoding” does NOT refer to the genetic code. Instead, the term“encoding” means that the user possesses a legend, key, or code, thatcorrelates each of many thousands of short nucleic acid sequences with asingle bead-bound compound.

A dramatic variation of using a bead that bears bead-bound compounds andbead-bound nucleic acids, where the nucleic acids encode the associatedcompounds, is as follows. The dramatic variation is to manufacture alibrary of conjugates, where each member of the library takes the formof a conjugate of a small molecule plus a DNA moiety, where the DNAmoiety encodes the small molecule). This conjugate is soluble and is notbead-bound. After screening with a cell or with a purified protein, theconjugate remains bound to the cell or purified protein, therebyenabling isolation of the conjugate and eventual identifying thecompound by sequencing the conjugated nucleic acid (see, Satz et al(2015) Bioconjugate Chemistry. 26:1623-1632).

Here, as in most of this patent document, the term “encode” does notrefer to the genetic code, but instead it refers to the fact that theresearcher uses a specific nucleic acid sequence to indicate a specific,known structure of a compound that is attached to it.

As an alternative to using an encoding strategy, such as the use of aDNA barcode, a bead that screens positive (thereby indicating a compoundthat screens positive) can be subjected to Edman degradation or to massspectrometry to identify the bead-bound compound (see, Shih et al (2017)Mol. Cancer Ther. 16:1212-1223). If the bead-bound compounds arepeptides, then MALDI mass spectrometry can be used for directdetermination of the sequence of a positively-screening peptidecompound. Direct sequencing is possible, because simultaneous cleavageand ionization occur under laser irradiation (Song, Lam (2003) J. Am.Chem. Soc. 125:6180-6188).

One fine point in performing split-and-pool synthesis of a combinatoriallibrary, is that the compound can be manufactured so that all of thecompounds share a common motif. This strategy has been described as the,“generation of a library of motifs rather than a library of compounds”(see, Sepetov et al (1995) Proc. Natl. Acad. Sci. 92:5426-5430; Lam etal, supra, at 418).

To provide a typical example of a large bead, the bead can be 0.1 mm indiameter and it can hold about 10¹³ copies of the same compound (Lam etal, supra). Following preparation of a library of bead-bound compounds,each bead can be used in individual assays, where the assays measurebiochemical activity or, alternatively, a binding activity. Assays canbe “on-bead” assays or, alternatively, the compound can be severed fromthe bead and used in solution-phase assays (Lam et al, supra).

Parameters of any type of bead include its tendency to swell in a givenassay medium, whether the bead's polymer is hydrophobic or hydrophilic,the identity of the attachments sites on the bead for attaching eachcompound, the issue of whether a spacer such as polyethylene glycol(PEG) is used to provide some separation of each compound from thebead's surface, and the internal volume of the bead.

Regarding the need to attach compounds to the bead, but at a distancefar away from the bead's hydrophobic surface, Lam et al, supra,discloses that polyoxyethylene-grafted styrene (TentaGel®) has theadvantage that the functionalizable group is at the end of apolyoxyethylene chain, and thus far away from the hydrophobicpolystyrene. Beads that possess a water-soluble linker include TentaGeland polydimethylacrylamide bead (PepSyn® gel, Cambridge ResearchBiochemicals, Northwitch, UK).

The parameter of internal volume can provide an advantage, where thereis a need to prevent interactions between the bead-bound DNA barcode andthe target of the bead-bound compound. To exploit this advantage, thebead can be manufactured so that the DNA barcode is situated in theinside of the bead while, in contrast, the compound that is beingscreened is attached to the bead's surface (Lam et al, supra, at pages438-439). This advantage of internal volume may be irrelevant, where thebead-bound compound is attached by a cleavable linker, and where assaysof the compound are conducted only on compounds that are cleaved andreleased.

Appell et al, provide a non-limiting example of spit-and-pool method forsynthesizing a chemical library followed by screening to detect activecompounds (Appell et al (1996) J. Biomolecular Screening. 1:27-31).Library beads are placed, one into each well, in an array of wells on afirst microwell plate, nanowell plate, or picowell plate. Beads areexposed to light, in order to cleave about 50% of the bead-boundcompounds, releasing them into solution in the well. Released compoundis then transferred to a second microwell plate, and subjected to assaysfor detecting wells that contain active compounds, thereby identifyingwhich beads in the first plate contain bead-bound compounds that areactive. Then, “[o]nce an active [compound] is identified from a singlebead, the bead is recovered and decoded, thus yielding the synthetichistory and . . . structure of the active compound” (Appell et al,supra).

For cell-based screening assays that screen for bead-bound compounds,Shih et al provide a novel type of bead (Shih et al (2017) Mol. CancerTher. 16:1212-1223). This novel type of bead contains a bead-boundcompound that is a member of a library of “synthetic death ligandsagainst ovarian cancer.” The bead is also decorated with biotin, wheretwo more chemicals are added that create a sandwich, and where thesandwich maintains adhesion of the cell to the bead. The sandwichincludes a streptavidin plus biotin-LXY30 complex. This sandwichconnects the bead to LXY30's receptor, which happens to be a well-knownprotein on the cell surface, namely, an integrin. The method of Shih etal, supra, resulted in the discovery of a new molecule (“LLS2”) that cankill cancer cells. The above method uses bead-bound compounds, where thecompounds bind to cells (even though the compound is still bead-bound).Cho et al created a similar one-bead-one-compound library, where thecompound being screened was sufficient to bind to cells (without anyneed for the above-described sandwich) (Cho et al (2013) ACSCombinatorial Science. 15:393-400). The goal of the Cho et al, reportwas to discover RGD-containing peptides that bind to integrin that isexpressed by cancer cells. The above-disclosed reagents and methods areuseful for the present disclosure.

Coupling Nucleic Acids to Beads (Orthogonal Style; Concatenated Style)

One way to get oriented to the topic of concatenated barcodes andorthogonal barcodes, is to note advantages that one has over the other.An advantage of orthogonal barcoding over concatenated barcoding, is asfollows. With attachment of each monomer of a growing chemical compound,what is attached in parallel is a DNA barcode module. With concatenatedbarcoding, if attachment of any given module is imperfect (meaning, thatnot all of the attachments sites was successfully coupled with a neededmodule), then the sequence of the completed barcode will not be correct.The statement “not be correct” means that imperfect coupling means thatchunks may be missing from wad was assumed to be the completed, correctDNA barcode. Here, the completed barcode sequence will contain amistake, due to failure of attachment of all of the modules. Incontrast, with orthogonal barcoding each individual module getscovalently bound to its own unique attachment site on the bead. Andwhere once a module gets attached to a given site on the bead, nofurther modules will be connected to the module that is alreadyattached.

The present disclosure provides reagents and methods for reducing damageto bead-bound DNA barcodes, and for reducing damage to partiallysynthesized bead-bound DNA barcodes. Each DNA barcode module, prior toattaching to a growing bead-bound DNA barcode, can take the form ofdouble stranded DNA (dsDNA), where this dsDNA is treated with a DNAcross-linker such as mitomycin-C. After completion of the synthesis ofthe DNA barcode in its dsDNA form, this dsDNA is converted to ssDNA.Conversion of dsDNA to ssDNA can be effected where one of the DNAstrands has a uracil (U) residue, and where cleavage of the DNA at theposition of the uracil residue is catalyzed by uracil-N-glycosidase(see, FIG. 5 of Ser. No. 62/562,905, filed Sep. 25, 2017. Ser. No.62/562,905 is incorporated herein by reference in its entirety). Theabove refers to damage that is inflicted on the growing DNA barcode byreagents used to make the bead-bound chemical compound.

Another method for reducing damage to bead-bound DNA barcodes, and forreducing damage to partially synthesized DNA barcodes, is bysynthesizing the DNA barcode in a double stranded DNA form, where eachof the DNA barcode modules that are being attached to each other takesthe form of dsDNA, and where each of the two strands is stabilized byway of a DNA headpiece. For eventual sequencing of the completed DNAbarcode, one of the strands is cleaved off from the DNA headpiece andremoved. The above refers to damage that is inflicted on the growing DNAbarcode by reagents used to make the bead-bound chemical compound (wherethis chemical compound is a member of the chemical library).

Yet another method for reducing damage to bead-bound DNA barcodes, is tosynthesize the DNA barcode in a way that self-assembles to form ahairpin, and where this DNA barcode self-assembles to that the firstprong of the hairpin anneals to the second prong of the hairpin.

Where the DNA barcode being synthesized takes the form of doublestranded DNA (dsDNA), solvents such as DCM, DMF, and DMA can denaturethe DNA barcode. The above methods and reagents can preventdenaturation.

As stated above, the term “DNA barcode” can refer to a polynucleotidethat identifies a chemical compound in its entirety while, in contrast,“DNA barcode module” can refer to only one of the monomers that make upthe chemical compound.

Another method for reducing damage to bead-bound DNA barcodes, and forreducing damage to partially synthesized DNA barcodes, is to use doublestranded DNA (dsDNA) and to seal the ends of this dsDNA by way of7-aza-dATP and dGTP.

In alternate embodiments, the method can use an intermediate between“concatenated DNA barcoding” and “orthogonal DNA barcoding,” where thisintermediate involves blocks of DNA barcodes, that is, where each blockcontains two DNA modules, or contains three DNA modules, or containsfour DNA modules, or contains five DNA modules, and the like (but doesnot contain all of the DNA modules that identify the full-lengthcompound).

FIG. 1 discloses an exemplary and non-limiting diagram of theCONCATENATED structured bead. The bead contains a plurality of DNAbarcodes (each made of DNA barcode modules) and a plurality of compounds(each made of chemical library monomers). For ease in speaking, the term“DNA barcode” may be used to refer to the polymer that includes all ofthe nucleic acids that are a “DNA barcode module,” as well as all of thenucleic acids that provide some function. The function can be anannealing site for a sequencing primer, or the function can be used toidentify a step in chemical synthesis of the bead-bound compound. FIG. 1also shows bead-bound compounds, where each compound is made of severalchemical library members, and where each chemical library member isrepresented by a square, circle, or triangle. FIG. 1 shows that each DNAbarcode module is numbered, consecutively, from 1 to 8, where thesenumbers correspond to the respective eight shapes (squares, circles,triangles). For clarity, nucleic acids that serve a function (and do notrepresent or “encode” any particular chemical unit) are not shown in thefigure.

FIG. 2 discloses an exemplary and non-limiting embodiment of theORTHOGONAL structured bead. The bead contains a plurality of DNAbarcodes (each made of DNA barcode modules), but each DNA barcode moduleis attached to a separate linking site on the bead. The entire DNAbarcode consists of eight DNA barcode modules, which in the figure arenumbered 1-8. When the information from a particular DNA barcode isread, and then used to identify the chemical compound that is bound tothe same bead, one must perform DNA sequencing on each of the separatelyattached DNA barcode modules. In FIG. 2, the bead also contains aplurality of attached chemical compounds, each with eight units, asshown by the eight shapes (circles, squares, triangles).

In FIG. 2, for clarity, functional nucleic acids that are attached toeach DNA barcode module is not shown. Of course, each of the DNA barcodemodules needs to have a nucleic acid that identifies the position of thechemical library monomer in the completed, full-length compound. For theexample shown in FIG. 2, the position needs to be first, second, third,fourth, fifth, sixth, seventh, or eighth.

In one embodiment, the chemical monomer is first attached and then,after that, the corresponding DNA barcode module is attached. In analternative embodiment, the DNA barcode module is first attached, andthen the corresponding chemical monomer is attached. Also, a procedureof organic synthesis can be followed that sometimes uses the “oneembodiment” and sometimes uses the “alternative embodiment.” In yetanother alternative embodiment, the present method provides block-wiseaddition of a block of several chemical monomers which is attached tothe bead, in parallel with attachment of a block of several DNA barcodemodules.

In exclusionary embodiments, what can be excluded is reagents,compositions, and methods that used block-wise addition of chemicalmonomers, of DNA barcode modules, or of both chemical monomers and DNAbarcode modules, to a bead.

This concerns nucleic acids that may be present in the bead-boundpolynucleotide, including nucleic acids that “encode” or serve toidentify monomers of a bead-bound compound. In exclusionary embodiments,the present disclosure can exclude a nucleic acid that encodes a“step-specific DNA sequencing primer site.” In this situation, for eachchemical monomer that is present in a compound, there is a correspondingDNA barcode module, where each DNA barcode module is flanked by at leastone corresponding primer-binding site, that is, “a step-specific DNAsequencing primer site.” Also, what can be excluded is a nucleic acidthat encodes or designates a particular step in the chemical synthesisof a compound, such as step 1, step 2, step 3, or step 4.

Moreover, the present disclosure can include a nucleic acid thatfunctions as a spacer. For example, as spacer can create a distance,along a polynucleotide chain, between a first site that is a sequencingprimer annealing site and a second site that identifies a chemicalmonomer. Also, the present disclosure can use a nucleic acid thatreiterates or confirms the information provided by another nucleic acid.Also, the present disclosure can use a nucleic acid that encodes a PCRprimer binding site. A PCR primer binding site can be distinguished froma sequencing primer, because a polynucleotide with a PCR primer bindingsite has two PCR primer binding sites, and because both of these sitesare designed to have the same melting point (melting point when the PCRprimer is annealed to PCR primer binding site).

In exclusionary embodiments, the present disclosure can exclude anucleic acid that functions as a spacer, or solely as a spacer. Also,the present disclosure can exclude a nucleic acid that reiterates orconfirms the info provided by another nucleic acid. Moreover, thepresent disclosure can exclude a nucleic acid that serves as a PCRprimer binding site, and can exclude a nucleic acid that serves as abinding site for a primer that is not a PCR primer.

Additionally, the present disclosure can exclude a nucleic acid thatidentifies the date that a chemical library was made, or that identifiesa step in chemical synthesis of a particular compound, or that serves asa primer annealing sequence.

Dedication of Sequencing Primers to a Particular DNA Barcode Module.

The present disclosure provides a DNA barcode that contains DNA barcodemodules and one or more sequencing primer annealing sites. Each DNAbarcode module may have its own, dedicated, sequencing primer bindingsite. Alternatively, one particular sequencing primer binding site maybe used for sequencing two, three, four, five, 6, 7, 8, 9, 10, or moreconsecutive DNA barcode modules, as may exist on the bead-bound DNAbarcode.

The following describes the situation where each DNA barcode module hasits own dedicated sequencing primer binding site. The present disclosureprovides a bead-bound concatenated barcode comprising a primer bindingsite capable of binding a DNA sequencing primer, wherein said primerbinding site is capable of directing sequencing of one or more of the1^(st) DNA barcode module, the 2^(nd) DNA barcode module, the 3^(rd) DNAbarcode module, the 4^(th) DNA barcode module, the 5^(th) DNA barcodemodule, and the 6^(th) DNA barcode module, and wherein the primerbinding site is situated 3-prime to the 1st DNA barcode module with noother DNA barcode module in between the 1^(st) DNA barcode module andthe primer binding site, 3-prime to the 2^(nd) DNA barcode module withno other DNA barcode module in between, 3-prime to the 3^(rd) DNAbarcode module with no other DNA barcode module in between, 3-prime tothe 4^(th) DNA barcode module with no other DNA barcode module inbetween, 3-prime to the 5^(th) DNA barcode module with no other DNAbarcode module in between, or 3-prime to the 6^(th) DNA barcode modulewith no other DNA barcode module in between.

Encoding Sequences and Sequences Complementary to Encoding Sequences.

The present disclosure can encompass any one, any combination of, or allof the encoding sequences disclosed above, or elsewhere, in thisdocument. In exclusionary embodiments, what can be excluded are any one,any combination of, or all of the encoding sequences disclosed above, orelsewhere, in this document. What can also be included or can beexcluded are double stranded nucleic acids that encode any one, anycombination of, or all of the encoding sequences described above, orelsewhere, in this document.

Orthogonal-Style DNA Barcode (Each DNA Barcode Module Attached toSeparate Location on Bead)

Synthesis of Orthogonal-Style Bead.

With orthogonal synthesis, each DNA module gets covalently attached to aseparate site on the bead, and where the result is that the entire DNAbarcode is contributed by a plurality of DNA modules. Where the DNAbarcode has the orthogonal structure, none of the DNA barcode modulesare attached to each other—instead each and every one of the DNA barcodemolecules has its own bead-attachment site that is dedicated to thatparticular DNA barcode module.

Nucleic acid identifying the synthesis step number for each DNA barcodemodule. In embodiments, the orthogonal DNA barcode includes a shortnucleic acid that identifies the first step of compound synthesis. Forthis embodiment, with the parallel attachment of the first chemicalmonomer and the first DNA barcode module, the first DNA barcode moduleactually takes the form of this complex of two nucleic acids: [SHORTNUCLEIC ACID THAT MEANS “STEP ONE” ] connected to [FIRST DNA BARCODEMODULE]. All of the nucleotides of this complex are in-frame with eachother and can be read in a sequencing assay, but the first short nucleicacid may optionally be attached to the first DNA barcode module by wayof a spacer nucleic acid.

The following continues the above description of the orthogonal DNAbarcode. The orthogonal DNA barcode includes a short nucleic acid thatidentifies the second step of compound synthesis. For this embodiment,with the parallel attachment of the second chemical monomer and thesecond DNA barcode module, the second DNA barcode module actually takesthe form of this complex of two nucleic acids: [SHORT NUCLEIC ACID THATMEANS “STEP TWO” ] connected to [SECOND DNA BARCODE MODULE]. All of thenucleotides of this complex are in-frame with each other and can be readin a sequencing assay, but the second short nucleic acid may optionallybe attached to the second DNA barcode module by way of a spacer nucleicacid.

The above-described method is repeated for the third, fourth, fifth,sixth, seventh, eighth, ninth, tenth, and up to the last of the DNAbarcode modules and up to the last of the chemical monomers, for anygiven bead. The above-method can be followed when using split-and-poolsynthesis, for creating DNA barcodes and chemical compounds that arebead-bound.

The orthogonal structure provides the following advantage over theconcatenated structure. With concatenated synthesis (all DNA barcodemodules attached to each other in one, continuous polymer) it is thecase that failure to achieve synthesis any of the intermediates couplingsteps can ruin the meaning of the concatenated DNA barcode that iseventually completed. In contrast, with orthogonal synthesis (each andevery one of the DNA barcode modules attached to a dedicated site on thebead), failure to attach any of the DNA barcode modules will only resultin an empty attachment site on the bead, and will not ruin the meaningof any of the other attached DNA barcode modules. In a preferredembodiment, each attached DNA barcode module includes an attached,second nucleic acid, where this second nucleic acid identifies the step(the step during the parallel synthesis of DNA barcode and chemicalcompound).

For orthogonal synthesis, it is acceptable for all of the attachmentsites on the bead to be used up (sites for attaching the growingchemical library member). However, for orthogonal synthesis, thechemical reaction needs to be designed so that the entire population ofattachment sites on the bead is only partly used up, with attachment ofthe first of many DNA barcode modules. The following provides optionallimits for using up sites during chemical synthesis of an orthogonalbarcode. For the non-modified bead, the total number of sites availablefor attaching a DNA barcode module is 100%.

Extent of using up attachment sites on a given bead, with synthesis ofan orthogonal-configured bead (regarding the 1^(st) DNA barcode). Thefollowing concerns attaching the first DNA barcode module. Inembodiments, with attachment of the first DNA barcode module, about 5%,about 10%, about 20%, about 30%, about 40%, or about 50% of the DNAbarcode attachments sites on the bead are used up. In other embodiments,less than about 2%, less than about 5%, less than about 10%, less thanabout 20%, less than about 30%, less than about 40%, or less than about50% of the DNA barcode attachments sites on the bead are used up. Instill other embodiments, with attachment of the first DNA barcodemodule, between 2-4%, between 2-6%, between 2-8%, between 2-10%, between2-12%, between 2-14%, between 2-16%, between 2-18%, between 2-20%,between 10-20%, between 10-25%, between 10-30%, between 10-35%, between10-40%, of the DNA barcode attachment sites are used up.

Regarding limits, with attaching the last of the DNA barcode modulesthat make up a particular DNA barcode, less than 20% of the sites areused up, less than 30%, less than 40%, less than 50%, less than 60%,less than 70%, less than 80%, less than 90%, less than 95%, or less than98% of the sites are used up.

Exclusionary embodiments can exclude beads or methods that match any ofthe above values or ranges. Also, exclusionary embodiments can excludebeads or methods that fail to match any of the above values or ranges.

The following concerns polymers that comprises one or more nucleicacids, each being a DNA barcode, as well as polymers that comprise twoor more nucleic acids, where some of the nucleic acids have abiochemical function such as serving as a primer-annealing site or as aspacer, and where other nucleic acids have an informational function andare DNA barcodes. In exclusionary embodiments, the present disclosurecan exclude a DNA barcode that includes a DNA crosslinking agent such aspsoralen. Also, what can be excluded is a DNA barcode with a primerbinding region with a higher melting temperature (or a lower meltingtemperature) than a DNA barcode module. This temperature can be merely“higher” or “lower” or it can be at least 2 degrees C. higher, at least4 degrees C. higher, at least 6 degrees C. higher, at least 8 degreeshigher, or at least 2 degrees C. lower, at least 4 degrees C. lower, atleast 6 degrees C. lower, at least 8 degrees lower.

Also what can be excluded is a method for making a DNA barcode that usesDNA ligase. Also, what can be excluded is a DNA barcode and methods formaking, that comprise a hairpin (ssDNA bent in a loop, so that oneportion of the ssDNA hybridizes to another portion of the same ssDNA).Additionally, what can be excluded is a composition with a nucleic acidhairpin, where the nucleic acid hairpin is covalently closed, forexample, with a chemical linker. Moreover, what can be excluded is a DNAbarcode that is covalently linked, either directly to a “headpiece,” orindirectly to “headpiece” (indirectly by way of covalent binding to oneor more chemicals that reside in between DNA barcode and the headpiece).

In other exclusionary embodiments, what can be excluded is a bead-boundDNA barcode, where the completed DNA barcode does not comprise anydouble stranded DNA (dsDNA), but only comprises single stranded DNA(ssDNA).

Extent of using up attachment sites on a given bead, with synthesis ofan orthogonal-configured bead (regarding the 2^(nd) DNA barcode). Thefollowing concerns attaching the second DNA barcode module. Inembodiments, with attachment of the second DNA barcode module (for thecreation of the orthogonal configured bead), about 5%, about 10%, about20%, about 30%, about 40%, or about 50% of the remaining free DNAbarcode attachments sites on the bead are used up. In other embodiments,less than about 5%, less than about 10%, less than about 20%, less thanabout 30%, less than about 40%, or less than about 50% of the remainingfree DNA barcode attachments sites on the bead are used up. In stillother embodiments, with attachment of the first DNA barcode module,between 2-4%, between 2-6%, between 2-8%, between 2-10%, between 2-12%,between 2-14%, between 2-16%, between 2-18%, between 2-20%, between10-20%, between 10-25%, between 10-30%, between 10-35%, between 10-40%,of the remaining free DNA barcode attachment sites are used up.

Exclusionary embodiments can exclude beads or methods that match any ofthe above values or ranges. Also, exclusionary embodiments can excludebeads or methods that fail to match any of the above values or ranges.

The above embodiments, as well as the above exclusionary embodiments,can also be applied to a method with attaching a third DNA modulebarcode, or with attaching a fourth DNA module barcode, or withattaching a fifth DNA barcode module, and so on.

Concatenated-Style DNA Barcode (all DNA Barcode Modules Reside in OneChain or Polymer, where the Entire Chain or Polymer is Attached to OneLocation on the Bead).

Synthesis of Bead-Bound Concatenated-Style DNA Barcode.

The present disclosure provides a bead-bound concatenated-style DNAbarcode, where the bead contains a plurality of concatenated-style DNAbarcodes, and where most or nearly all of the plurality ofconcatenated-style DNA barcodes have essentially the same structure. Theconcatenated-style DNA barcode can contain one or more DNA barcodemodules, where the ordering of these DNA barcode modules (from thebead-attachment terminus to the distal terminus) along the entire DNAbarcode, takes the same order as the time that the bead-boundconcatenated-style DNA barcode is synthesized. Also, the ordering ofthese DNA barcode modules along the entire DNA barcode, takes the sameorder as the time that a corresponding chemical library monomer iscoupled to the growing bead-bound compound.

The concatenated-style DNA barcode can comprise, in this order, a linkerthat is used to couple the entire concatenated-style DNA barcode to thebead. Also, it can comprise, in this order, a 1^(st) DNA barcode module,a 1^(st) annealing site, a 2^(nd) DNA barcode module, a 2^(nd) annealingsite, a 3^(rd) DNA barcode module, and a 3^(rd) annealing site.

One Ordering of Sequencing Primer Hybridizing Site in a Bead-Bound DNABarcode.

In sequencing primer hybridizing site embodiments, theconcatenated-style DNA barcode can comprise, in this order, a linker, a1^(st) DNA barcode module, a 1^(st) annealing site, a 1^(st) sequencingprimer binding site, a 2^(nd) DNA barcode module, a 2^(nd) annealingsite, a 2^(nd) sequencing primer binding site, a 3^(rd) DNA barcodemodule, a 3^(rd) annealing site, and a 3^(rd) sequencing primer bindingsite, and so on.

Another Ordering of the Sequencing Primer Hybridizing Site, as it Occursin a Bead-Bound DNA Barcode.

In another sequencing primer hybridizing site embodiment, theconcatenated-style DNA barcode can comprise, in this order, a linker, a1^(st) DNA barcode module, a 1^(st) sequencing primer binding site,1^(st) annealing site, a 2^(nd) DNA barcode module, a 2^(nd) sequencingprimer binding site, a 2^(nd) annealing site, a 3^(rd) DNA barcodemodule, a 3^(rd) sequencing primer binding site, and 3^(rd) annealingsite, and so on.

The Term “Annealing Site.”

The term “annealing site” is used to refer to an annealing site that ispart of a splint oligonucleotide (splint oligo) and also to refer to thecorresponding bead-bound annealing site that resides on a growingbead-bound DNA barcode. The skilled artisan understands that the“annealing site” on the splint oligo does not possess the same DNAsequence as the corresponding “annealing site” on the growing bead-boundDNA barcode. In other words, the skilled artisan understands that onesequence is complementary to the other sequence. Therefore, it is of noconsequence that, for the descriptions herein, both annealing sites havethe same name. In other words, it is of no consequence that the 2^(nd)annealing site on a splint oligo is disclosed as one that hybridizes tothe 2^(nd) annealing site on growing bead-bound DNA barcode.

Synthesis in Blocks.

In an alternative embodiment, the growing compound and the growingsequence of DNA barcode modules can be synthesized in blocks. Forexample, a block consisting of 2-chemical library units can be attachedto a bead in parallel with attaching a block consisting of corresponding2-DNA barcode modules. Similarly, a block consisting of 3-chemicallibrary units, can be attached to a bead in parallel with attaching ablock consisting of a corresponding 3-DNA barcodes. Block synthesisinvolving blocks of four, blocks of five, blocks of six, blocks ofseven, blocks of eight, blocks of nine, blocks of ten, and so on, arealso provided. Each of these block transfer embodiments can also beexcluded by the present disclosure. The blockwise transfer of DNAbarcode monomers can be done orthogonally, with unique attachment pointsfor receiving each of successive blocks of DNA barcode monomers.Alternatively, blockwise transfer of DNA barcode monomers can be done toproduce a concatemer structure (all DNA barcode modules occurring asonly one continuous, linear polymer).

Also, during split-and-pool synthesis in parallel of the bead-bound DNAbarcode and the bead-bound compound, synthesis of can occur in blocks.The block can take the form of two or more chemical library monomers,and the block can take the form of two or more DNA barcode modules.

Location of Split-and-Pool Synthesis.

Split-and-pool synthesis can be used for the parallel synthesis ofbead-bound compounds and bead-bound concatenated DNA barcode. Also,split-and-pool synthesis can be used for the parallel synthesis ofbead-bound compounds and bead-bound orthogonal DNA barcode. Theconcatenated DNA barcode can be made by way of the “splint oligo”method. Alternatively, concatenated DNA barcode can be made by way ofclick chemistry. Also, a combination of the “splint oligo” method andclick chemistry can be used. Split-and-pool synthesis can occur in a 96well plate, where each well has a floor made of a 0.25 micrometerfilter. Under normal gravity conditions, aqueous solutions do not flowthrough this filter. However, suction can be applied to remove anyaqueous solutions from all of the 96 wells, for example, where there isa need to replace a first aqueous solution with a second aqueoussolution. This suction method is used when the bead is exposed to afirst set of reagents, or when the first set of reagents needs to berinsed out, or when the first set of reagents needs to be replaced by asecond set of reagents. A manifold is used to hold the 96 well plate(Resprep VM-96 manifold) and a pump can be used to draw fluid out thebottom of every filter (BUCHI Vac V-500 pump). The 96 well plate withthe filter bottom was, AcroPrep Advance 96 well, 350 uL, 0.45 um, REF8048 (Pall Corp., Multi-Well Plates, Ann Arbor, Mich.).

Distance from Primer Annealing Site to a DNA Barcode Module.

For the purpose of sequencing a bead-bound DNA barcode, that is, for thegoal of sequencing all of the DNA barcode modules that form the DNAbarcode, a polynucleotide comprising a first nucleic acid that is anannealing site for a sequencing primer, and a second nucleic acid thatis a DNA barcode module, the first nucleic acid can be immediatelyupstream of the second nucleic acid. Alternatively, the first nucleicacid can be upstream of the second nucleic acid, where the first andsecond nucleic acids are separated from each other by one, two, three,four, five, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides, orby about one, about two, about three, about about four, about five,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, or about 15 nucleotides. The separation can be withnucleic acids that merely serve as a spacer or, alternatively, theseparation can be with a third nucleic acid that encodes information,such as step number in a multi-step pathway of organic synthesis, or thenume of a class of chemical compounds, or a disease that might betreatable by the bead-bound compound, or the date, or a lot number, andso on.

Synthesis of Bead-Bound Concatenated DNA Barcode Using Click Chemistry

Click chemistry can be used for the step-by-step synthesis of a DNAbarcode. Here, what can be coupled is a first DNA barcode moduledirectly to a bead, or a first DNA barcode module to a bead-boundlinker.

Also, what can be coupled is a polynucleotide taking the form of a firstnucleic acid that is a 1^(st) DNA barcode module attached to a secondnucleic acid that is a 1^(st) sequencing primer binding site. Thissequencing primer binding site allows the operator to determine thesequence of the 1^(st) DNA barcode module.

To provide another example, what can be coupled is a 2^(nd) DNA barcodemodule directly to a bead-bound 1^(st) DNA barcode module.Alternatively, what can be coupled is a polynucleotide taking the formof a first nucleic acid that is a 2^(nd) DNA barcode module attached toa second nucleic acid that is a 2^(nd) sequencing primer binding site.This sequencing primer binding site allows the operator to determine thesequence of the 2^(nd) DNA barcode module. If there is read-through tothe 1^(st) DNA barcode module, then what can be determined is thesequence of both of these DNA barcode modules.

To provide yet another example, what can be coupled is a polynucleotidecomprising a first nucleic acid that is a 1^(st) DNA barcode module, anda second nucleic acid that identifies the step in a multi-step parallelsynthesis of the DNA barcode and of the compound. Also, oralternatively, the second nucleic acid can identify the general class ofcompounds that are being made by the split-and-pool synthesis. Also, oralternatively, the second nucleic acid can identify a disease that is tobe treated by the compounds to be screened. Also, the second nucleicacid can identify the date, or the name of the chemist, and so on.

A preferred method for synthesizing the DNA barcode is shown below,where the same cycle of reactions is used with progressive attachment ofeach DNA barcode module.

Step 1.

Provide a bead with an attached TCO group. In actual practice, the beadwill have hundreds or thousands of identically attached TCO groups,where each TCO group is attached to a different site on the bead. Also,in actual practice, a large number of beads will be simultaneouslymodified by click chemistry, with employment of the split-and-poolmethod.

Step 2.

Add [tetrazine]-[first DNA barcode module]-[azide] to the bead, andallow the TCO group condense with the tetrazine group. The result is thefollowing construct: BEAD-TCO-tetrazine-first DNA barcode module-azide.In actual practice, this construct does not include any TCO ortetrazine, but instead has the condensation product that is created whenTCO condenses with tetrazine.

Step 3.

Optional wash.

Step 4.

Add DBCO-TCO in order to cap the azide and to create a TCO terminus. Theresult is the following structure:

BEAD-TCO-tetrazine-first DNA barcode module-azide-DBCO-TCO

Step 5.

Optional wash.

Step 6.

Add the following reagent, which attaches the second DNA barcode module.Attachment is to the distal terminus of the growing DNA barcode. Thereagent is:

[tetrazine]-[second DNA barcode module]-[azide] to the bead, and allowthe TCO group condense with the tetrazine group. The result is thefollowing construct:

BEAD-TCO-tetrazine-first DNA barcodemodule-azide-DBCO-TCO-[tetrazine]-[second DNA barcode module]-[azide]

The above scheme includes a cycle of steps for the stepwise addition ofmore and more DNA barcode modules, where these additions are in parallelwith additions of more and more chemical monomers. As stated elsewhere,this “parallel” synthesis can involve attaching a chemical monomerfollowed by attaching a DNA barcode module that identifies that monomeror, alternatively, attaching a DNA barcode module followed by attachinga chemical monomer that is identified by that particular chemicalmonomer.

Compounds for Click-Chemistry Synthesis of DNA Barcode

FIG. 17 discloses the chemical synthesis of a compound suitable forconnecting a deoxycytidine reside (dC) during the synthesis of a DNAbarcode module and, ultimately, the entire DNA barcode. The startingmaterial is N4-acetyl-2′-deoxy-5′-O-DMT cytidine. The abbreviation “DMT”stands for 4,4-dimethoxytrityl. The final product of this multi-steppathway of organic synthesis bears a cytosine moiety, a triphosphategroup, and a propargyl group that is attached to the 3′-position of theribose group. The propargyl group is used for click chemistry, where itcondenses with an azide group to produce a covalent bond. Aftercondensing, the result is that a residual chemical (never naturallypresent in nucleic acids), occurs as a “scar” from the click chemistrythat had been performed. What is available is DNA polymerases that canbe used for sequencing-by-synthesis of DNA barcodes made by clickchemistry, and where the DNA polymerases can move across the scars, andwhere the scars do not cause sequencing errors. TBAI is tetrabutylammonium iodide.

Synthesis of Concatenated Configuration DNA Barcode

In the following description, DNA barcode modules are assembled in a rowin order to create the DNA barcode. However, in the in-text diagramsthat are shown below, the term “DNA barcode” is used instead of “DNAbarcode module,” in order to make the in-text diagrams fit on the page.FIG. 7 illustrates the same steps as shown here, but with additionaldetails, such as diagrams of beads. A reiterated sequence of reactionscan be used for adding each additional DNA barcode module.

Option of Creating a DNA Barcode that Includes a Terminal Nucleic Acidthat Encodes DNA Hairpin.

This concerns a DNA barcode that includes, at the 3-prime end, a nucleicacid that possesses an annealing site for a sequencing primer, a bendtaking the form of about four bases that are not base-paired, and asequencing primer that is capable of bending around and forming basepairs with the sequencing primer annealing site. To repeat, thesequencing primer anneals to the sequencing primer annealing site, wherethe actual sequencing reaction begins at the 3′-terminus of the annealedsequencing primer.

When it is time to perform a final step in synthesizing a DNA barcode,and when the final DNA barcode module is to be coupled to the growingbead-bound DNA barcode, the “splint oligo” can include a sequence thatencompasses a DNA hairpin (the DNA hairpin including, in this order, anannealing site for the sequencing primer, several nucleotides that donot base pair with each other or with any nearby sequences of bases, anda sequencing primer). After annealing the “splint oligo,” then DNApolymerase and dNTPs are added, where polymerization occurs at the3′-end of the growing DNA barcode, where what gets polymerized using thesplint oligo as a template is, in order: (1) Annealing site forsequencing primer; (2) Bend in the hairpin taking the form of four orfive deoxyribonucleotides that do not base pair with teach other; and(3) Sequencing primer.

Reversible Terminator Group at the 3′-End of the Hairpin SequencingPrimer.

The present disclosure provides reagents, compositions, and methods, forattaching a pre-formed complex of a nucleotide/reversible terminatorgroup, to the 3′-terminus of the annealed sequencing primer. Reversibleterminator group is an optional component of the hairpin sequencingprimer, where it is to be part of a bead-bound DNA barcode.

Step 1.

At the start, we have a bead situated in a picowell, where the beadbears a coupled polynucleotide, and where the 5′-end of thepolynucleotide is coupled to the bead, optionally, with a linker. FIG. 7shows that the bead-bound polynucleotide comprises a 1^(st) DNA barcodeand a 1^(st) annealing site. The linker can be made of a nucleic acid,or it can be made of some other chemically. Preferrably, the linker ishydrophobic, and preferably the linker separates the bead-bound DNAbarcode from the hydrophobic polystyrene bead, for example, a TentaGel®bead.

For convenience in writing, a 1^(st) annealing site that is part of abead-bound DNA barcode and a 1^(st) annealing site that is part of asoluble “splint oligo” are both called “1^(st) annealing site,” eventhough they do not have the same sequence of bases (instead, thesequence of bases are complementary to each other, where the result isthat the splint oligo can hybridize to the 1^(st) annealing site on thebead-bound growing DNA barcode, thereby serving as a template for DNApolymerase to extend the bead-bound DNA barcode by copying what is onthe splint oligo.

Also, for convenience in writing, a 2^(nd) annealing site that is partof a bead-bound DNA barcode and a 2^(nd) annealing site that is part ofa soluble “splint oligo” are both called, “2^(nd) annealing site,” eventhough they do not have the same sequence (but instead havecomplementary bases).

The bead-bound growing DNA barcode, from the 5′-end to the 3′-end, maycontain the nucleic acids in the following order:

Bead-/1^(st) DNA Barcode/1^(st) Annealing Site/

Alternately, the bead-bound growing DNA barcode, from the 5′-end to the3′-end, may include a nucleic acid that encodes the step number, wherethe bead-bound growing DNA barcode has nucleic acids in the followingorder:

Bead-/1^(st) DNA Barcode/Nucleic Acid Encoding Step Number/1^(st)Annealing Site/

Alternatively, the bead-bound growing DNA barcode can include a nucleicacid that is a functional nucleic acid (a sequencing primer annealingsite), as shown below:

Bead-/1^(st) DNA Barcode/Sequencing Primer Annealing Site/1^(st)Annealing Site/

What is not shown in these in-text diagrams is an optional linker thatmediates coupling of the DNA barcode to the bead. The linker can takethe form of a nucleic acid, or it can be made of some other organicchemical.

Step 2.

Add a soluble splint oligonucleotide (splint oligo), where this splintoligo comprises a 1st annealing site and a 2^(nd) DNA barcode module,and a 2^(nd) annealing site.

FIG. 7 also illustrates the step where the hybridized splint oligo isused as a template, where DNA polymerase catalyzes the attachment to thebead-bound growing DNA barcode of the 2^(nd) DNA barcode module and the2^(nd) annealing site. FIG. 7 shows the enzymatic product where DNApolymerase catalyzes uses the splint oligo as a template, resulting inthe bead-bound DNA barcode growing by a bit longer (growing by covalentattachment of the 2^(nd) DNA barcode and the 2^(nd) annealing site. Whatis shown immediately below in the text, is the complex of the splintoligo that is hybridized to the bead-bound growing DNA barcode:

Bead-/1^(st) DNA Barcode/1^(st) Annealing Site/

. . . 1^(st) Annealing Site/2^(nd) DNA Barcode/2^(nd) Annealing Site

To reiterate some information shown in FIG. 7, what is shown immediatelybelow is the splint oligo:

“1^(st) Annealing Site/2^(nd) DNA Barcode/2^(nd) Annealing Site”

Step 3.

DNA polymerase and dNTPs are added to extend the bead-bound DNA barcode.Shown below is the bead-bound growing DNA barcode, with the splint oligostill hybridized to it, and where the bead-bound growing barcode islonger than before, because what is now attached to it is a nucleic acidthat is the “2^(nd) DNA barcode module” and a nucleic acid that is the“2^(nd) annealing site.” FIG. 7 also illustrates this step. The splintoligo is shown underneath the bead-bound growing barcode:

Bead-/1^(st) DNA Barcode/1^(st) Annealing Site/2^(nd) DNA Barcode/2^(nd)Annealing Site

. . . 1^(st) annealing site/2^(nd) DNA barcode/2^(nd) Annealing Site

Step 4.

Wash away the splint oligo. The splint oligo can be encouraged todissociate from the bead-bound growing barcode by heating, that is, byheating the entire picowell plate, for example, to about 60 degrees C.,about 65 degrees C., about 70 degrees C., about 75 degrees C., about 80degrees C., for about ten minutes or, alternatively, by adding diluteNaOH to the picowell array, and then neutralizing.

Step 5.

Add a second splint oligo which, after hybridizing to the bead-boundgrowing splint oligo, can be used as a template for mediating DNApolymerase-catalyzed attachment of a 3^(rd) DNA barcode and a 3^(rd)annealing site. This second splint oligo, which is a soluble reagent, isshown below (but it is not shown in FIG. 7):

2^(nd) Annealing Site/3^(rd) DNA Barcode/3^(rd) Annealing Site/

Step 6.

Allow this oligonucleotide to anneal to the corresponding bead-bound“2^(nd) annealing site,” and allow DNA polymerase to extend thebead-bound oligonucleotide, so that it contains a complement to the:“3^(rd) DNA barcode/3^(rd) annealing site/

Step 7.

Wash away the second splint oligo.

Step 4.

Add the following splint oligo (this particular addition is not shown inFIG. 7).

3rd Annealing Site/4th DNA Barcode/4th Annealing Site/

This soluble oligonucleotide has a nucleic acid that can anneal to the“3^(rd) annealing site” of the bead-bound oligonucleotide. Onceannealed, DNA polymerase with four dNTPs are employed and used forextending the bead-bound oligonucleotide to encode yet another DNAbarcode module (the 4^(th) DNA barcode). The above cycle of steps isrepeated, during the entire split-and-pool procedure that creates, inparallel, the library of chemical compounds and the associated DNAbarcodes, where each DNA barcode is associated with a given compound(where each DNA barcode informs us of the history of chemical synthesisof the associated compound). The above cycle of steps is stopped, whenthe chemical synthesis of the library of compounds has been completed.With the completed bead-bound, DNA barcoded chemical library in hand,the beads can then be dispensed into microwells of a microwell array.

The DNA barcode for each bead also constitutes a DNA barcode thatassociated with each microwell. The DNA barcode allows identification ofthe bead-bound compound. The sequencing method of the present disclosureoccurs inside the microwell while the bead is still inside themicrowell. In exclusionary embodiments, the present disclosure canexclude any sequencing method and can exclude any reagents used forsequencing, where sequencing is not performed on a DNA template that isbead-bound, or where sequencing is not performed on a bead-bound DNAtemplate that is situated inside a microwell.

Annealing Sites for Sequencing Primer.

In one embodiment, each DNA barcode module in a completed DNA barcode isoperably linked and in frame with its own sequencing primer annealingsite, thus providing the operator with the ability to conduct separatesequencing procedures on each DNA barcode module (in this embodiment, itis preferred that each DNA barcode module is also operably linked withits own nucleic acid that identifies (encodes) the step in synthesis ofthe entire DNA barcode.

In another embodiment, each DNA barcode has only one sequencing primerannealing site, where this can be situated at or near the 3′-terminus ofthe bead-bound DNA barcode, and where the sequencing primer itself canbe soluble, added to the picowell, and then hybridized to the sequencingprimer annealing site. Alternatively, where the sequencing primer is tobe part of a DNA hairpin, this DNA hairpin is added by way of a “splintoligo” at the final step in creating the bead-bound DNA barcode. FIG. 7does not show any annealing sites for any sequencing primers.

Nucleic Acids Coupled to Beads by Way of the 3′-Terminus of the NucleicAcid

While various embodiments disclosed in this invention pertain tocoupling DNA to a bead by way of the DNA's 5′-end, in other embodiments,DNA such as a DNA barcode or a DNA tag, can be coupled to the bead byway of their 3′-end. The 3′-hydroxyl group of DNA might be reactiveunder certain chemical synthesis conditions (e.g. Mitsunobutransformations), rendering the 3′-end damaged and unable to participatein extension, ligation or other steps. Thus DNA tags may be attached tobeads via their 3′-ends to prevent unwanted chemical reactions and toprevent damage to the DNA barcodes.

Exclusionary Embodiments Regarding Bead-Bound DNA Barcodes of thePresent Disclosure.

What can be excluded is any bead, microparticle, microsphere, resin, orpolymeric composition of matter, wherein the concatenated DNA barcode islinked to the bead by way of a photocleavable linker or by way of acleavable linker.

What can be excluded is any bead, microparticle, microsphere, resin, orpolymeric composition of matter, that does not include both of thefollowing: (1) Concatenated DNA barcode that is coupled to a firstposition on the bead, (2) A compound that is coupled to a secondposition on the bead, and wherein the first position is not the same asthe second position. In a preferred embodiment, this “compound” is madeof a plurality of chemical library monomers.

What can be excluded is any bead, microparticle, microsphere, resin, orpolymeric composition of matter, that does not have an exterior surface(or exterior surfaces) and also an interior surface (or interiorsurfaces, or interior regions), and where the bead does not comprise atleast 10,000 substantially identical concatenated DNA barcodes that arecoupled to the bead, and wherein at least 90% of the at least 10,000substantially identical concatenated DNA barcodes are coupled to theexterior surface. In other words, what can be excluded is any bead whereat least 90% of the coupled concatenated DNA barcodes are not coupled tothe exterior surface.

What can be excluded is any bead, microparticle, microsphere, resin, orpolymeric composition of matter, that is made substantially ofpolyacrylamide or that contains any polyacrylamide.

What can be excluded is any bead, microparticle, microsphere, hydrogel,resin, or polymeric composition of matter, that contains a promoter,such as a T7 promoter, or that contains a polyA region, or that containsa promoter and also a polyA region.

Method with Only One Cycle of Annealing/Polymerization, to Produce aBead-Bound DNA Barcode with Two DNA Barcode Modules.

The present disclosure encompasses systems, reagents, and methods, wherethe bead-bound DNA barcode includes only one annealing/polymerizationstep. This embodiment is represented by the following diagrams, wherethe first diagram shows annealing of the splint oligo, and the seconddiagram shows filling-in using DNA polymerase. The end-result is abead-bound DNA barcode that contains two DNA barcode modules. In thisparticular procedure, the bead-bound starting material can optionallyinclude linker (but preferably not any cleavable linker), optionally anucleic acid that encodes information other than identifying a chemicalcompound, and optionally a functional nucleic acid, such as a sequencingprimer or a DNA hairpin. The two diagrams are shown in the text (see,immediately below):

Bead-/1^(st) DNA Barcode/1^(st) Annealing Site/

. . . 1^(st) Annealing Site/2^(nd) DNA Barcode/2^(nd) Annealing Site

Bead-/1^(st) DNA Barcode/1^(st) Annealing Site/2^(nd) DNA Barcode/2^(nd)Annealing site

. . . 1^(st) Annealing Site/2^(nd) DNA Barcode/2^(nd) Annealing Site

Method with Two Cycles of Annealing/Polymerization, to Produce aBead-Bound DNA Barcode that has Three DNA Barcode Modules.

The present disclosure encompasses bead-bound compositions, systems, andmethods, where two different split oligos are used (first splint oligo;second splint oligo). In this situation, the first splint oligocomprises the structure: 1^(st) annealing site/2^(nd) DNA barcode/2^(nd)annealing site, and where the second splint oligo comprises thestructure: 2^(nd) annealing site/3^(rd) DNA barcode/3^(rd) annealingsite.

Method with Three Cycles of Annealing/Polymerization, to Produce aBead-Bound DNA Barcode that has Four DNA Barcode Modules.

The present disclosure encompasses bead-bound compositions, systems, andmethods, where three different split oligos are used (first splintoligo; second splint oligo, third splint oligo). In this situation, thefirst splint oligo comprises the structure: 1^(st) annealing site/2^(nd)DNA barcode/2^(nd) annealing site, and where the second splint oligocomprises the structure: 2^(nd) annealing site/3^(rd) DNA barcode/3^(rd)annealing site, and where the third splint oligo comprises thestructure: 3^(rd) annealing site/4^(th) DNA barcode/4^(th) annealingsite.

Method with Four Cycles of Annealing/Polymerization, to Produce aBead-Bound DNA Barcode that has Five DNA Barcode Modules.

The present disclosure encompasses bead-bound compositions, systems, andmethods, where four different split oligos are used (first splint oligo;second splint oligo, third splint oligo, fourth splint oligo). In thissituation, the first splint oligo comprises the structure: 1^(st)annealing site/2^(nd) DNA barcode/2^(nd) annealing site, and where thesecond splint oligo comprises the structure: 2^(nd) annealingsite/3^(rd) DNA barcode/3rd annealing site, and where the third splintoligo comprises the structure: 3^(rd) annealing site/4^(th) DNAbarcode/4^(th) annealing site, and where the fourth splint oligocomprises the structure: 4^(th) annealing site/5^(th) DNA barcode/5^(th)annealing site,

Embodiments with a Plurality of Steps of Annealing/Polymerization, toProduce a Bead-Bound DNA Barcode that has a Plurality of DNA BarcodeModules.

The present disclosure encompasses bead-bound compositions, systems, andmethods, relating to concatenated barcodes, that uses only one splintoligo (making a 2-module DNA barcode), that uses only two splint oligos(making a 3-module DNA barcode), that uses only three splint oligos(making a 4-module DNA barcode), that uses only four splint oligos(making a 5-module DNA barcode), that uses only five splint oligos(making a 6-module DNA barcode), that uses only six splint oligos(making a 7-module DNA barcode), and so on.

What is encompassed is bead-bound compositions, systems, and methods,that uses at least one splint oligo, at least two splint oligos, atleast three splint oligos, at least four splint oligos, at least fivesplint oligos, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at last 13, at least 14, at least 20splint oligos, or less than 20, less than 15, less than 10, less than 8,less than 6, less than 4, less than 3, less than 2 splint oligos. Thesenumbers refer to the splint oligo itself, as well as to the number ofthe step of adding the splint oligo, and also to the numbering of theDNA module that is added to the growing bead-bound DNA barcode.

Reducing Damage to DNA Barcodes

Reducing Damage by Using Orthogonal DNA Barcodes (Instead ofConcatenated DNA Barcodes).

One way to get oriented to the topic of concatenated DNA barcodes andorthogonal DNA barcodes, is to note advantages that one has over theother. An advantage of orthogonal barcoding over concatenated barcoding,is as follows. With attachment of each monomer of a growing chemicalcompound, what is attached in parallel are chemical library monomers tocreate a chemical library, and DNA barcode modules to create a completedand full-length DNA barcode.

With concatenated barcoding, if attachment of any given module isimperfect (meaning, that not all of the attachments sites weresuccessfully coupled with a needed module), then the sequence of thecompleted barcode will not be correct. The statement “not be correct”means that imperfect coupling resulted in chunks that were missing,where the user had assumed that the completed product was a completedand correct DNA barcode. Here, the completed DNA barcode sequence willcontain a mistake, due to failure of attachment of all of the DNAmodules. In contrast, with orthogonal barcoding each individual DNAmodule gets covalently bound to its own unique attachment site on thebead. And where once a DNA module gets attached to a given site on thebead, no further DNA modules need to get coupled to the DNA modules thatare already coupled to the bead.

Reducing Damage by Using Cross-Linkers.

The present disclosure provides reagents and methods for reducing damageto bead-bound DNA barcodes, and for reducing damage to to partiallysynthesized bead-bound DNA barcodes. Each DNA barcode module, prior toattaching to a growing bead-bound DNA barcode, can take the form ofdouble stranded DNA (dsDNA), where this dsDNA is treated with a DNAcross-linker such as mitomycin-C. After completion of the synthesis ofthe DNA barcode in its dsDNA form, this dsDNA is converted to ssDNA.Conversion of dsDNA to ssDNA can be effected where one of the DNAstrands has a uracil (U) residue, and where cleavage of the DNA at theposition of the uracil residue is catalyzed by uracil-N-glycosidase(see, FIG. 5 of Ser. No. 62/562,905, filed Sep. 25, 2017. Ser. No.62/562,905 is incorporated herein by reference in its entirety). Theabove refers to damage that is inflicted on the growing DNA barcode byreagents used to make the bead-bound chemical compound.

Reducing Damage by Using Double Stranded DNA (dsDNA) for Making DNABarcodes.

Another method for reducing damage to bead-bound DNA barcodes, and forreducing damage to partially synthesized DNA barcodes, is bysynthesizing the DNA barcode in a double stranded DNA form, where eachof the DNA barcode modules that are being attached to each other takesthe form of dsDNA, and where each of the two strands is stabilized byway of a DNA headpiece. For eventual sequencing of the completed DNAbarcode, one of the strands is cleaved off from the DNA headpiece andremoved. The above refers to damage that is inflicted on the growing DNAbarcode by reagents used to make the bead-bound chemical compound (wherethis chemical compound is a member of the chemical library).

Reducing Damage by Including a Hairpin.

Yet another method for reducing damage to bead-bound DNA barcodes, is tosynthesize the DNA barcode in a way that self-assembles to form ahairpin, and where this DNA barcode self-assembles to that the firstprong of the hairpin anneals to the second prong of the hairpin.

Where the DNA barcode being synthesized takes the form of doublestranded DNA (dsDNA), solvents such as DCM, DMF, and DMA can denaturethe DNA barcode. The above methods and reagents can preventdenaturation.

Reducing Damage by Using Sealed Ends of dsDNA.

Another method for reducing damage to bead-bound DNA barcodes, and forreducing damage to partially synthesized DNA barcodes, is to use doublestranded DNA (dsDNA) and to seal the ends of this dsDNA by way of7-aza-dATP and dGTP.

Reducing Damage by Avoiding Proteic Solvents, Avoiding Strong Acids andBasis, Avoiding Strong Reducing Agents and Oxidants.

The type of chemistry that is compatible with the presence ofdeoxyribonucleic acids (DNA), whether bead-bound DNA or DNA that is notbead-bound, may require absence of proteic solvents, avoiding strongacidic conditions, avoiding strong basis such as t-butyl lithium,avoiding strong reducing agents such as lithium aluminum hydride,avoiding reagents that react with DNA bases, such as some alkyl halides,and avoiding some oxidants (see, Luk and Sats (2014) DNA-CompatibleChemistry (Chapter 4) in A Handbook for DNA-Encoded Chemistry, 1^(st)ed. John Wiley and Sons, Inc.).

As stated elsewhere, the term “DNA barcode” can refer to apolynucleotide that identifies a chemical compound in its entiretywhile, in contrast, “DNA barcode module” can refer to only one of themonomers that make up the chemical compound.

Reducing Damage to Nucleic Acids by Using DNA-Compatible Chemistry.

Satz et al, disclose various chemistries that are compatible withbead-bound nucleic acids (Satz et al (2015) Bioconjugate Chemistry.26:1623-1632; correction in Satz et al (2016) Bioconjugate Chem.27:2580-2580). Although the descriptions in Satz et al, supra, concernchemical reactions that are performed on DNA/chemical library memberconjugates, the types of DNA-compatible chemistries that are describedare also relevant, where the organic chemistry is to be performed on abead that contains bead-bound compounds and bead-bound DNA.

DNA-compatible reactions for the formation of benzimidazole compounds,imidazolidinone compounds, quinazolinone compounds, isoindolinonecompounds, thiazole compounds, and imidazopyridine compounds aredisclosed (see, Satz et al, Table 1, entries 1-6).

Moreover, DNA-compatible protecting groups are disclosed as including,alloc deprotection, BOC deprotection, t-butyl ester hydrolysis,methyl/ethyl ester hydrolysis, and nitro reduction with hydrazine andRaney nickel (see, Satz et al, Table 1, entries 7-11).

Furthermore, methods for coupling reagents to DNA are disclosed, wherethe coupling occurs with a functional group that is already attached tothe DNA. The methods include Suzuki coupling, an optimized procedure forthe Sonogashira coupling between an alkyne and an arylhalide, theconversion of aldehydes to alkynes usingdimethyl-1-diazo-2-oxopropylphosphonate, a new method for triazole cycloaddition directly from purified alkyne, an improved method for reactionof isocyanate building blocks with an amine functionalized DNA where theimproved reaction occurs with isocyanate reagent at pH 9.4 buffer (see,Satz, et al, Table 1, entries 12-15).

Additional methods for coupling reagents to DNA are disclosed, where thecoupling occurs to a functional group already attached to the DNA. Theseinclude a method where a primary amine is conjugated to DNA, anoptimized procedure to form DNA-conjugated thioureas, a method toalkylate secondary amines and the bis-alkylation of aliphatic primaryamides, monoalkylation of a primary amine DNA-conjugate, usinghetarylhalides as building blocks that can be reacted withamine-functionalized DNA-conjugate, and methods for Wittig reactions(see, Satz et al, Table 1, entries 16-20).

Reducing Damaged DNA by Way of DNA Repair Enzymes.

Various proteins, including enzymes, DNA-damage binding proteins, andhelicases, are available for repairing DNA damage. What is commerciallyavailable is DNA repair proteins that can repair oxidative damage,radiation-induced damage, UV light-induced damage, damage fromformaldehyde adducts, and damage taking the form of alkyl group adducts.Glycoside enzyme, which remove damaged bases (but do not cleave ssDNA ordsDNA) are available to repair 5-formyluracil, deoxyuridine, and5-hydroxymethyluracil. T4PDG is available to repair pyrimidine dimers.hNEIL1 as well as Fpg are available to repair oxidized pyrimidines,oxidized purines, apurinic sites, and apyrimidinic sites. EndoVIII isavailable to repair oxidized pyrimidine and apyrimidinic sites. EndoV isavailable for repairing mismatches. HaaG is a glycosylase that isavailable for repairing alkylated purines. Where a DNA repair enzymeleaves a gap, where double stranded DNA has a gap where one or morecontinuous deoxyribonucleotides are missing in one of the strands, thenvarious DNA polymerases are available for filling in the gap (see,Catalog (2018) New England BioLabs, Ipswich, Mass.).

A variety of DNA repair enzymes and DNA repair systems have beenisolated from mammals, yeast, and bacteria. These include those thatmediate nucleotide excision repair (NER), direct repair, base excisionrepair, transcription-coupled DNA repair, and recombinational repair.Interstrand DNA crosslinks can be repaired by combined use of NER andhomologous recombination. Direct repair includes repair of cyclobutanepyrimidine dimers and 6-4 products, by way of photolyase enzymes. Directrepair also includes removal of O⁶-methyl from O⁶-methylguanine by DNAmethyltarnsferase. See, Sancar et al (2004) Ann. Rev. Biochem. 73:39-85;Hu, Sancar (2017) J. Biol. Chem. 292:15588-15597.

The present disclosure provides systems, reagents, and methods forrepairing damage to bead-bound DNA barcodes by treating with a DNArepair enzyme, or by a complex of DNA repair proteins, and the like.

Reducing Damage Via Coupling DNA to Beads Via their 3′-End.

Certain chemical transformation may damage exposed 3′-hydroxyl groups ofnucleic acids. For instance Mitsunobu reactions allow the conversion ofprimary and secondary alcohols to esters, phenyl ethers, thioethers andvarious other compounds, which might render exposed 3′-ends unreactiveto subsequent processing steps, or cause the now modified 3′-end toparticipate in further chemical reactions. In some embodiments, the DNAtags may be attached to beads via their 3′-end, so only the 5′-end isexposed to solution.

The reagents, systems, and methods of the present disclosure encompassbead-bound nucleic acids, such as a bead-bound DNA or a bead-bound DNAtags, where coupling to the bead involves the 3′-terminus (or the3′-end) of the DNA. Where ssDNA that comprises a DNA barcode is coupledby way of the 3′-end, of the ssDNA, sequencing can be initiated byhybridizing only one sequencing primer, where this sequencing primerhybridizes upstream of the entire DNA barcode, and where thishybridizing is at or near the bead-bound end of the coupled ssDNA. As analternative to using only one sequencing primer, a plurality ofsequencing primers can be used, where each sequencing primer hybridizesupstream to a particular DNA barcode module. For example, if a given DNAbarcode contains five DNA barcode modules, and where the DNA is coupledto a bead by way of its 3′-end, the DNA barcode can include fivedifferent primer annealing sites, where each primer annealing site islocated just upstream, or immediately upstream, of a given DNA barcodemodule.

Double Stranded DNA (dsDNA) Coupling Embodiments.

In other embodiments, what is coupled to the bead is dsDNA, where the3′-terminus of only one of the strands in the dsDNA are coupled to thebead. In a 5′-coupling embodiment that involves dsDNA, what can becoupled is dsDNA, where the 5′-terminus of only one of the strands ofthe dsDNA is coupled to the bead.

(V) Coupling Chemical Compounds to Beads

The present disclosure provides: (1) Linkers to attach chemical librarymember to a substrate, such as a bead; (2) Linkers to attach nucleicacid barcode to a substrate, such as a bead; (3) Cleavable linkers, forexample, cleavable by UV light, cleavable by an enzyme such as aprotease; (4) Non-cleavable linkers; (5) Bifunctional linkers; (6)Multi-functional linkers; and (7) Plurality of beads used for linking.Available, for example, is 4-hydroxymethyl benzoic acid (HMBA) linker,4-hydroxymethylphenylacetic acid linker (see, Camperi, Marani, Cascone(2005) Tetrahedron Letters. 46:1561-1564).

A “non-cleavable linker” may be characterized as a linker that cannot bedetectably cleaved by any reagent, condition, or environment, that isused during the steps of a given organic chemistry procedure.Alternatively, a “non-cleavable linker” may be characterized as a linkerthat cannot be cleaved, except by a reagent, condition, or environmentthat is unacceptably destructive towards other reactants, products, orreagents of a given organic chemistry procedure.

A bifunctional linker, or other multifunctional linker, can take theform of a fork (fork used by humans for consuming food), where thehandle of the fork is attached to a bead, and where each tine of thefork are linked to one of a variety of chemicals. For example, one tinecan be linked to a chemical library member. Another tine can be linkedto a DNA barcode. Yet another tine of the fork can be linked to a metalion.

Regarding use of a multiplicity of beads, the present disclosureprovides multiple-bead embodiments, such as: (1) A first bead containingattached nucleic acid barcode linked to a second bead, where the secondbead contains attached chemical library member; (2) A first beadcontaining an attached nucleic acid barcode linked to a second bead,where the second bead contains an attached chemical library member, andwhere a third bead is attached (to one or both of the first bead andsecond bead), and where the third bead contains a covalently attachedreagent. The attached reagent can be an enzyme, where the enzyme is usedfor assaying activity of the attached chemical library member.

(VI) Coupling Monomers Together to Make a Compound

Exemplary Chemical Monomers.

Amino acid derivatives suitable for use as chemical monomers for thecompositions and methods of the present disclosure are shown in FIG. 4.The figure indicates a source of the chemicals, for example, AnaSpec EGTGroup, Fremont, Calif.; Sigman-Aldrich, St. Louis, Mo.; Acros Organics(part of ThermoFisher Scientific), or Combi-Blocks, San Diego, Calif.

Additional chemical monomers are shown in FIGS. 22-27. Each of FIGS.22-27 provides the structure, chemical name, and an associated DNAmodule barcode. As disclosed on the figures, compounds 1-6 (FIG. 22),the respective barcodes are ACGT, ACTC, AGAC, AGCG, AGTA, and ATAT. Forcompounds 7-10 (FIG. 23), the respective barcodes are, ATGA, CACG, CAGC,and CATA. For compounds 11-16 (FIG. 24), the respective barcodes are,CGAG, CGCT, CGTC, CTAC, CTGT, and GACT. For compounds 17-21 (FIG. 25),the respective barcodes are GAGA, GCAC, GCTG, GTAG, and GTCA. Forcompounds 22-26 (FIG. 26), the respective barcodes are GTGC, TAGT, TATC,TCAG, and TCGC. And for compounds 27-30 (FIG. 27), the respectivebarcodes are TCTA, TGAT, TGCA, and TGTG. These barcodes are onlyexemplary. For any given library of compounds, a different collection ofDNA barcodes may be used to identify each of the chemical monomers thatare used to build the compounds in that library.

Coupling Reactions.

The following describes coupling chemical monomers to the bead and toeach other, that is, where a first step is coupling the first chemicalmonomer directly to the bead by way of a cleavable linker, and wheresubsequent chemical monomers are then connected to each other, one byone. The conditions disclosed below are DNA compatible.

This Describes Methods to Make Three Amino Acid Compounds on Tentagel®Beads.

The Fmoc protected resin (1 mg, Rapp polymere GmbH, 10 um, TentaGelM-NH2, 0.23 mmol/g) modified with Fmoc-Photo-Linker,4-{4-[1-(9-Fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy} butanoic acid) or another appropriatelinker with Fmoc protection was suspended inside each well of a reactorplate (Merck Millipore Ltd, 0.45 um hydrophobic PTFE) in DMA (150 uL).The solvent was removed by application of a vacuum to the bottom of theplate with a Resprep VM-96 vacuum manifold. The Fmoc protecting groupwas removed by suspending the resin in 150 uL of a mixture of 5%piperazine, 2% DBU in DMF. The plate was sealed with an Excel ScientificAlumna Seal and shaken at 40 C for 15 min. The solvent was removed by anapplied vacuum and the deprotection procedure repeated for 5 min. Afterfiltration each well was washed with 150 uL each of 2×DMA, 3×DCM, 1×DMAwith a vacuum applied between each wash to remove the solvent. Each wellof resin was then acylated by the appropriate amino acid by adding 150uL of a pre-activated mixture of 60 mM Fmoc-amino acid, 80 mM Oxyma, 200mM DIC and 80 mM 2,4,6-trimethylpyridine that was allowed to sit for 2min at room temperature. The plate was again sealed and shaken for 1 hrat 40 degrees C. After filtration each well was washed with 150 uL eachof 2×DMA and 3×DCM. The beads in each well were re-suspended in 150 ulof DCM and each well's contents combined through pipetting into a singlereceptacle. The combined beads are thoroughly mixed and redistributedinto the plate through pipetting equal amounts in the appropriate wells(1 mg/well). The solvent was removed by an applied vacuum and each wellwas ready for the next appropriate step. For each additional amino acidcoupling, first the Fmoc deprotection step is repeated followed by thecoupling step with the desired amino acid. If a split and pool isrequired, the combining and redistribution method is repeated.

This Describes a Method for Creating 3-Mer Amino Acid by Split-PoolMethod on Beads.

The Fmoc protected resin (1 mg, Rapp polymere GmbH, 10 um, TentaGelM-NH₂, 0.23 mmol/g) modified with Fmoc-Photo-Linker,4-{4-[1-(9-Fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy} butanoic acid) or any other appropriatelinker was suspended inside each well of a reactor plate (MerckMillipore Ltd, 0.45 um hydrophobic PTFE) in DMA (150 uL). The solventwas removed by application of a vacuum to the bottom of the plate with aResprep® VM-96 vacuum manifold. The Fmoc protecting group was removed bysuspending the resin in 150 uL of a mixture of 5% piperazine, 2% DBU inDMF. The plate was sealed with an Excel Scientific Alumna Seal andshaken at 40 C for 15 min. The solvent was removed by an applied vacuumand the deprotection procedure repeated for 5 min. After filtration eachwell was washed with 150 uL each of 2×DMA, 3×DCM, 1×DMA with a vacuumapplied between each wash to remove the solvent. Each well of resin wasthen acylated by the appropriate AA by adding 150 uL of a pre-activatedmixture of 60 mM Fmoc-amino acid, 80 mM Oxyma, 200 mM DIC and 80 mM2,4,6-trimethylpyridine that was allowed to sit for 2 min at roomtemperature. The plate was again sealed and shaken for 1 hr at 40 C.After filtration each well was washed with 150 uL each of 2×DMA, 3×DCM,1×DMA. For each additional AA coupling, first the Fmoc deprotection stepis repeated followed by the coupling step with the desired AA. Toanalyze each successive coupling, a 1 mg portion of beads was suspendedin 100 uL DMSO and exposed to full power of the 365 nm LED for twohours. The resin is filtered off and the filtrate injected onto anAgilent 1100 series LCMS equipped with a Agilent Poroshell SB-C-18,3.0×50 mm, 2.7 um column. A gradient of 5% CH3CN in 0.1% TFA in water to100 CH3CN in 0.1% TFA over 4 min at a flow rate of 1.2 mL/min andmonitored at 220 nm was ran.

Experiment to Make Non-Amino Acid Pendant with Lenalidomide (Revlimid®)Attached.

This would be attached to the last amino acid after deprotection. Thiswas also done in a spin. Each well of resin was acylated (after an Fmocdeprotection) with 150 uL of a 5 min preaged mixture of 40 mM chloroacetic acid, 40 mM Oxyma, 80 mM DIC, and 40 mM TMP in DMA. The plate wassealed and shaken at 40 C for 1 hr. Each well was washed with 150 uLeach of 3×DMA, 3×DCM, and 2×DMA. The resin was then re-suspended in asuspension of 100 mM K2CO3 and 100 mM Rev in DMA. The plate was sealedand shaken for 3 hrs at rt. The resin was washed with 150 uL each of2×50/50 DMA/water, 3×DMA, 3×DCM, and 2×DMA.

Defining the Degree of Fidelity of Synthesis of a Chemical Compound thatis attached to a given bead.

This concerns the completed chemical compound, where the chemicalcompound is a member of a chemical library. Each chemical compound maybe made, in part, or in full, from chemical monomers. The followingcharacterizes chemical compounds that are attached to a given bead. Thisgiven bead may be the product of split-and-pool based synthesis of alibrary of chemical compounds, where each bead possesses a uniquechemical compound.

Members of a chemical library can be synthesized on a solid support,such as on a bead, by way of solid phase synthesis. Solid phasesynthesis of chemicals with peptide bonds is characterized by use of onethe following two chemical groups. The first chemical group is,N-alpha-9-fluorenyl-methyloxycarbonyl (Fmoc, base labile). The secondchemical group is, tert-butyloxycarbonyl (tBoc, acid labile) (see,Vagner, Barany, Lam (1996) Proc. Natl. Acad. Sci. 93:8194-8199). Fmocand tBoc are protecting groups that can be used to protect peptidesubstrates, where the Fmoc group or tBoc group is attached to thealpha-amino group (Sigler, Fuller, Verlander (1983) Biopolymers.22:2157-2162).

Preferably, at least 99.5%, at least 99.0%, at least 95%, at least 90%,at least 85%, or at least 80% of the member of the chemical librarybound to a given bead, following completed synthesis, has exactly thesame chemical structure. It is possible that incomplete coupling thatmight occur at one or more steps in the multi-step synthesis of thechemical library member. For this reason, the compositions of thepresent disclosure may be characterized and limited by one of thefollowing limits or ranges.

What is also provided by the present disclosure are methods and reagentswhere at least 5%, at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, or at least 70%, or at least 80%, or atleast 90%, or at least 95%, or at least 99%, of the members of thechemical library bound to a given bead has, following completedsynthesis, exactly the same chemical structure (these numbers do takeinto account, and reflect, errors that might occur during solid phasesynthesis, for example, failure of one growing compound to receive oneof the chemical monomers. Also, these numbers do take into account, andreflect, chemical damage to any of the monomers that might occur duringsolid phase synthesis).

In exclusionary embodiments, the present disclosure can exclude anymethod or reagent that does not meet one of the above cut-off values for“exactly the same structure.”

In an alternate embodiment, two beads, 3 beads, 4 beads, 5 beads, about5-10 beads, about 10-20 beads, about 20-40 beads, about 40-80 beads, ina population of beads, contain the same and identical chemical compound(without taking into account any errors in incorporation of chemicalmonomers during solid phase synthesis, and without taking into accountany chemical damage that occurs to a chemical monomer during organicsynthesis).

Introduction to Click Chemistry.

According to Jewett et al, “Click reactions are defined . . . as thosethat . . . [are] selective, high yielding, and having good reactionkinetics. A subclass of click reactions whose components are inert tothe surrounding biological milieu is termed biorthogonal” (Jewett andBertozzi (2010) Chem. Soc. Rev. 39:1272-1279). “Click chemistry” can beused for joining small units together with heteroatom links, such ascarbon-X-carbon. Click chemistry can be used alone, or in conjunctionwith other types of chemical reactions, for the synthesis of drugs ordrug candidates. Click chemistry works well with procedures used forcombinatorial chemistry. Reactions in click chemistry are characterizedby high yields, by being irreversible, by insensitivity to oxygen orwater. Classes of chemical reactions used in “click chemistry” include:(1) Cycloaddition reactions, especially from the 1,3-dipolar family andfrom hetero-Diels Alder reactions; (2) Nucleophilic ring-openingreactions, as with strained heterocyclic molecules such as epoxides,aziridines, and cyclic sulfates; (4) Carbonyl chemistry of the non-aldoltype; and (5) Addition to carbon-carbon multiple bonds, as withoxidation reactions and some Michael addition reactions. Click chemistryreactions are distinguished by their high thermodynamic driving force,often greater than 20 kcal/mol while, in contrast, non-click chemistryreactions involve forming bonds with only a modest thermodynamic drivingforce (Kolb and Sharpless (2003) Drug Discovery Today. 8:1128-1137,Kolb, Finn, Sharpless (2001) Angew. Chem. Int. Ed. 40:2004-2021).

Tetrazine and Trans-Cyclooctene (TCO).

Tetrazine, such as, 1,2,4,5-tetrazine, can react with trans-cyclooctene(TCO) by way of a Diels-Alder cyclo addition (Devaraj, Haun, Weissleder(2009) Angew. Chem. Intl. 48:7013-7016).

Hartwig-Buchwald Amination.

Hartwig-Buchwald amination reactions can be used in the solid-phasesynthesis of pharmaceuticals. This amination reactions is used tosynthesize carbon-nitrogen bonds, where the reaction involves:aryl-halide plus amine (R₁—NH—R₂), as catalyzed by palladium, to producean aryl product where the amine replaces the halide, and where thenitrogen of the amino group is directly attached to the aromatic ring.The end-result is a product involving a carbon (of aryl group) tonitrogen (of amino group) bond. Stated another way, the reactionconverts arylhalides into the corresponding anilines. Hartwig-Buchwaldamination is compatible with a variety of amines, and is well-suited forcombinatorial chemistry (Zimmermann and Brase (2007) J. Comb. Chem.9:1114-1137).

Huisgen Cycloadditions.

Huisgen 1,3-dipolar cycloaddition reactions involve alkynes and organicazides. Alkynes have the structure, R—C═CH. Azides have the structure,R—N⁺═N═N⁻. Copper catalysts accelerate the rate of the Huisgencycloaddition reaction. The Huisgen reaction operates by way of “clickchemistry” or “click reactions.” Huisgen reaction, when catalyzed bycopper, can produce a 1,2,3-triazole nucleus suitable for making smallmolecule drugs. Huisgen reaction is compatible with the presence ofamino acid side chains, at least when in a protected form. Moleculesmade with a 1,2,3-triazole may possess a bond that is similar to theamide bonds of polypeptides, and thus these molecules can be a surrogatefor the peptide bond (Angell and Burgess (2007) Chem. Soc. Rev.36:1674-1689).

Peptide Nucleic Acids (PNAs).

The present disclosure provides the methods of split and pool chemistry,combinatorial chemistry, or solid phase chemistry, for synthesizingpeptide nucleic acids. Peptide nucleic acids are analogues ofoligonucleotides. They resist hydrolysis by nucleases. They can bindstrongly to their target RNA sequences. Uptake of peptide nucleic acidsinto cells can be enhanced by “cell penetrating peptides” (Turner,Ivanova, Gait (2005) Nucleic Acids Res. 33:6837-6849; Koppelhus (2008)Bioconjug. Chem. 19:1526-1534). Peptide nucleic acids can be made bysolid phase synthesis and by combinatorial synthesis (see, Quijano,Bahal, Glazer (2017) Yale J. Biology Medicine. 90:583-598; Domling(2006) Nucleosides Nucleotides. 17:1667-1670).

The present disclosure encompasses bead-bound compounds, where thecompound takes the form of only one monomer. For example, thisbead-bound compound can take the form of lenalidomide, or it can takethe form of lenalidomide with an attached carboxylic acid group, or aform of lenalidomide where the amino group has been modified with asmall chemical moiety that bears a carboxylic acid group, or where thecompound is a lenalidomide analog that is a stereoisomer or anenantiomer of lenalidomide.

(VII) Split and Pool Synthesis and Parallel Synthesis

This concerns use of the “split and pool” method for synthesizing alibrary of chemical compounds, and the method where the “split and“pool” method is used for the simultaneous synthesis of bead-boundchemical compounds and bead-bound DNA barcodes. This also describessplitting and pooling to make a mixed set of compounds. At a laterpoint, what is disclosed below is coupling of a non-amino acid, as wellas the preparations of beads that are modified by polyethylene glycol(PEG).

The present disclosure provides split and pool synthesis for generatingchemical libraries. In one embodiment, this method involves the steps:(a) Split beads into different containers; (b) Add a different buildingblock to each container. For example, where three container are used,add and react Species A to the first containing, Species B to the secondcontainer; and Species C to the third container, where the speciesbecome covalently bound to attachment sites on whatever bead is in thecontainer; (c) Pool all beads together in one container; (d) Split beadsinto three containers, (e) Add a different building block to eachcontainer, where Species A is added to the first container, Species B isadded to the second container, and Species C is added to the thirdcontainer, where the species become covalently bound to the firstspecies that had been previously attached (see, Stockwell (2000) TrendsBiotechnol. 18:449-455).

The split-and-pool synthesis of the present disclosure includes, eitherbefore or after each chemical coupling step (making the chemical librarymember), a DNA-barcode coupling step, where this DNA barcode identifiesthe chemical that is being coupled in that step.

In exclusionary embodiments, the present disclosure can exclude methodsand reagents where, for a given step of parallel synthesis, a barcode isattached prior to attaching a chemical. Conversely, the presentdisclosure can exclude methods and reagents where, for a given step ofparallel synthesis, a chemical is attached prior to attaching a barcode.

One characteristics of a bead-bound chemical library that is prepared bythe split and pool method, is that each bead will have only one type ofcompound attached to it. Where there is incomplete coupling, forexample, if for a given split and pool step, only 4,000 out of 5,000attachment sites was successfully coupled with the desired chemicalspecies, then some heterogeneity will occur.

Parallel Synthesis.

In a preferred embodiment of the present disclosure, parallel synthesiscan be used for organic synthesis of a chemical compound and of theassociated DNA barcode. In actual practice, modification of a bead byone more chemical monomers and modification of the same bead by one moreDNA barcode modules, is not strictly in parallel. In actual practice,the bead receives one more chemical unit (chemical monomer) followed byreceiving a DNA barcode module that encodes that particular chemicalunit. The term “parallel” refers to the fact that, as the polymer ofchemical library monomers grows, the polymer of DNA barcode module alsogrows. When all of the DNA barcode modules have been attached to thebead, to form either a CONCATENATED structure or an ORTHOGONALstructure, the full-length DNA barcode is called a “DNA barcode” (andnot merely a DNA barcode module).

Ratio of Number of Externally Attached DNA Barcode to Total Number ofAttached Chemical Library Member.

This concerns external surfaces and internal surfaces of a bead. For agiven bead that has externally attached DNA barcodes (without regard tonumber of internally attached DNA barcodes) and attached chemicallibrary member (attached to both external surface as well as to internalsurfaces), the ratio of number of externally attached DNA barcode numbertotal attached chemical library member number can be, for example, about0.1:100, about 0.2:100, about 0.5:100, about 1.0:100, about 2:100, about5:100, about 10:100, about 20:100, about 30:100, about 40:100, about50:100, about 60:100, about 70:100, about 80:100, about 90:100, about1:1, about 100:150, about 100:200; about 100:400; about 100:600, and thelike. In exclusionary embodiments, the present disclosure can excludeany bead, or any population of beads, that fits into one of the abovevalues.

Homogeneity of DNA Barcode for a Typical Bead; Homogeneity of ChemicalLibrary Member for a Typical Bead

The present disclosure provides, for any given bead (or for anypopulation of beads) a “chemical library homogeneity” that is at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least92%, at least 94%, at least 96%, at least 98%, at least 99.5%, and thelike.

In less stringent embodiment, the present disclosure provides, for anygiven bead or, alternatively, for any given population of beads, a“chemical library homogeneity that is at least 10%, at least 20%, atleast 30%, at least 40%, or at least 50%.

Similarly, the present disclosure provides the above cut-off values forassessing homogeneity of a barcode, such as a DNA barcode.

Homogeneity for DNA barcode and homogeneity for a chemical librarymember may be defined, in terms, of percent of total population thatconforms to the exact sequence as planned and desired by the methodssection of a lab manual or notebook.

In exclusionary embodiments, the present disclosure can exclude anyreagent, composition, or method, that does not conform with one or moreof the above cut-off values.

Where one assesses homogeneity of a population of beads, one needs toaccount homogeneity for the sum of bead #1, bead #2, bead #3, bead #4,bead #5, bead #6, bead #7, and so on, for the situation wherehomogeneity is desired throughout the entire population of beads.

In exclusionary embodiments, the present disclosure can exclude anybead, or any population of beads, where homogeneity of DNA barcode isnot at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 92%, at least 94%, at least 96%, at least 98%, at least99.5%, and the like. Also, in exclusionary embodiments, the presentdisclosure can exclude any bead, or any population of beads, wherehomogeneity of chemical library member is not at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 92%, at least94%, at least 96%, at least 98%, at least 99.5%, and the like.

Ratio of Internally Attached DNA Barcodes to Externally Attached DNABarcodes

In some embodiments of the present disclosure, it might be desired tomanufacture and use beads where DNA barcodes are mainly attached on theexterior surface. One reason to NOT make and use beads with internal DNAbarcodes, is the low permeation of DNA oligomers to the interior spaces,and low permeation of DNA ligases to interior spaces (ligases forconnecting DNA modules to each other to create the finished DNAbarcode). And for sequencing purposes, a reason to NOT make and useinternal DNA barcodes, is low permeation of enzymes needed to amplifyDNA needed for eventual sequencing of the barcode. Yet another reasonNOT to make and use beads with internal DNA barcodes is to fee upinterior space for attaching members of the chemical library.

The present disclosure provides beads bearing DNA barcodes, where theratio of internally attached DNA barcodes to externally attached DNAbarcodes is about 0.1:100, about 0.2:100, about 0.4:100, about 0.8:100,about 1:100, about 2:100, about 4:100, about 8:100, about 10:100, about20:100, about 40:100, about 50:100, about 60:100, about 70:100, about80:100, about 90:100, about 1:1, and so on.

Also, the present disclosure provides beads bearing DNA barcodes, wherethe ratio of internally attached DNA barcodes to externally attached DNAbarcodes is under 0.1:100, under 0.2:100, under 0.4:100, under 0.8:100,under 1:100, under 2:100, under 4:100, under 8:100, under 10:100, under20:100, under 40:100, under 50:100, under 60:100, under 70:100, under80:100, under 90:100, under 1:1, and so on.

A population of beads, in an aqueous suspension, can be contacted to asubstrate, such as a microwell array, resulting in beads entering andoccupying the microwells. The ratio of the number of beads in thesuspension to the number of microwells in the substrate can be adjusted,to arrive at a desired occupancy. For example, if the suspensioncontains only one bead, then every microwell that contains a bead willcontain only one bead, where the remaining microwells will not containany bead. If the suspension contains 20,000 beads and if the substratecontains 200,000 microwells, then at least 180,000 microwells will betotally empty of beads, and where most of the microwells that contain abead will contain only one bead. A small percentage of occupiedmicrowells will contain two beads.

In value embodiments, the ratio of bead number in the suspension tomicrowell number can be about 0.2:100, about 0.4:100, about 0.6:100,about 0.8:100, about 1:100, about 2:100, about 4:100, about 6:100, about8:100, about 10:100, about 20:100, about 30:100, about 40:100, about50:100, about 60:100; 80:100, about 100:100 (same as 1:1), about 2:1,about 4:1, about 6:1, about 8:1, about 10:1, and the like.

In exclusionary embodiments, the present disclosure can exclude anymethod or system, that falls into one of the above values or ranges.

In range embodiments, the ratio of bead number in the suspension tomicrowell number can be about 0.2:100 to about 0.4:100, about 0.4:100 toabout 0.6:100, about 0.6:100 to about 0.8:100, about 0.6:100 to about1:100, about 1:100 to about 2:100, about 2:100 to about 4:100, about4:100 to about 6:100, about 0.6:100 to about 8:100, about 8:100 to about10:100, about 10:100 to about 20:100, about 20:100 to about 30:100,about 30:100 to about 40:100, about 40:100 to about 50:100, about 50:100to about 60:100, about 60:100 to 80:100; about 80:100 to about 100:100(same as 1:1), about 100:100 (same as 1:1) to about 2:1, about 2:1 toabout 4:1, about 4:1 to about 6:1, about 6:1 to about 8:1, about 8:1 toabout 10:1, and the like.

In exclusionary embodiments, the present disclosure can exclude anymethod or system, that falls into one of the above values or ranges.

(VIII) Fabricating Picowells

Combination of UV Light, Photomask, and Photoresist for Manufacturing aPicowell Array Plate.

Plates that include many microwells or picowells can be fabricated asfollows for use in the present disclosure. In brief, a sandwich of threelayers is assembled. The top layer is photoresist. The middle layer is aglass wafer. The bottom layer is a photomask. The picowells will becarved out of the photoresist by UV light. After the picowells arecarved out of the flat sheet of photoresist, the photoresist resembles atypical metal pan that contains cups for baking muffins, and where thecups in the pan that are used for holding muffin batter have angledsides. The UV light acts as an “un-cross linker” because it breaks downthe photoresist's polymer. After UV treatment, solvent is added to washout the UV treated photoresist, leaving clean-looking picowells.

Rotating at an Angle to Create Angled Walls.

Picowells with angled walls are created as follows. The photomask hasmany apertures, where each aperture corresponds to the desired bottomdimension of the picowell. The bottom dimensions can include acircumference, diameter, and a shape, that is, a round shape. The topdimension of the well is created by directing an angled UV light towardsthe apertures in the photomask while rotating the light source orrotating the stage that holds the sandwich (photomask/glasswafer/photoresist sandwich). With rotation, the light source is not at a90 degree angle to the photomask/wafer/photoresist sandwich, but insteadis slightly angled away from the 90 degree point, in order to carve outangled walls in each picowell. The resulting picowell array plate thatcontains many picowells can be used as is. Alternatively, this picowellarray plate can be used as a mold for the inexpensive creating of manypicowell array plates.

Han et al describes equipment and reagents for manufacturing microwellplates where the microwells have angled walls (see, Han et al (2002) J.Semiconductor Technology and Science. 2:268-272). What is described is aUV source, a contact stage, a tilting stage, and the SU-8 photoresist.Fabrication begins with a single side polished silicon wafer. SU-8photoresist is coated on the wafer at about 0.10 to 0.15 mm thick. Then,the photoresist is soft baked on a 65 degrees C. hot plate for 10 minand then on a 95 degrees C. hot plate for 30 minutes. The resultingphotoresist/wafer sandwich is contacted with a UV mask using a contactstage. The term “Inclined and rotated UV lithography” refers to a methodfor manufacturing microwell array plates or picowell array plates, whereeach well has an angled wall. Here, the floor of the well has a smallerdiameter and the top of the well (where the top edge of the well meetsthe flat surface of the plate) has a wider diameter. For exposure withUV light, a turntable is used and where the UV light is inclined (Han etal, supra). The mask is contacted with the photoresist where each of theapertures in the mask are circular. FIG. 8 of Han et al, supra, providesa picture of the direction of UV light, the UV mask, the photoresiststructure, the wafer substrate, and the turntable. Han et al describeshow to manufacture a truncated cone. A soft material such as PDMS(polydimethylsiloxane) may be poured over the cone array and cured,whereupon peeling the PDMS layer, conical wells are formed.

Creating a Mold for Use in Mass-Production of Picowell Array Plates.

Where a picowell array plate has been manufactured, epoxy can be pouredover the plate resulting in filling all of the picowells and connectingall of the filled picowells with a platform of epoxy. Once the epoxy hassolidified, the solid platform with the attached array ofpicoprotuberances is removed (the picoprotuberance being the reverse ofthe desired picowell). The solid platform with picoprotuberances is areusable molding that can be used for the manufacture of many picowellarray plates.

The procedure for making replicates from the epoxy mold (or a cone arraymold made of any hard material is called, “hot embossing.” Briefly, asubstrate material is heated to its glass transition temperature orsoftening temperature, at which point the mold with picoprotrubances isuniformly pressed against the heat-softened material. The mold can beseparated from the substrate after the picoprotrubances are transferredas pico-invaginations into the substrate material. This disclosurepreferably discloses pico-cones and picowells as the patterns of themold and substrate, respectively.

Hot embossing, epoxy masters, and photoresist such as the SU-8photoresist are described (see, Bohl et al (2005) J. Micromechanics andMicroengineering. 15:1125-1130, Jeon et al (2011) Biomed Microdevices.13:325-333; Liu, Song, Zong (2014) J. Micromechanics andMicroengineering. 24:article ID:035009; del Campo and Greiner (2007) J.Micromechanics and Microengineering. 17:R81-R95).

Other Microwell Plate Embodiments.

Plastic microwell arrays can be manufactured by way of a thermal formingusing a silicon mold, where the silicon mold possesses an array ofmicrowells, for example, an array of 800,000 microwells. A high degreeof control that results in tapered geometries and smooth sidewalls, andsubmicron tolerances can be created with use of a non-pulsed dry etchprocess. In contrast, methods that use a pulsed dry etch process, suchas the Bosch process, can result in rough sidewalls and lack of controlover lateral dimensions during etching.

Using non-pulsed dry etch process, plastic arrays are fabricated bythermally forming plastic on a silicon master that is created by anon-pulsed isotropic dry etch process using a chrome mask. This processuses three gases, Ar, SF₆, and C₄F₈. The process is conducted at a RFpower between 1200 to 2000 Watts and a bias of 150 Watts. Fine-tuning ofthe taper of the silicon mold with production of smooth sidewalls can beaccomplished by varying the gas flow between the three gases. What isvaried is the ratio of SF₆ to C₄F₈, where the result of changing theratio is, for example, a tapered wall of the mold (the silicon pillar)that resides at an angle of 18 degrees (very slanted walls), 9 degrees(slightly slanted walls), or 2 degrees (walls almost perpendicular tosubstrate) (see, Perry, Henley, and Ramsey (Oct. 26-30, 2014)Development of Plastic Microwell Arrays for Improved ReplicationFidelity. 18^(th) Int. Conference on Miniaturized Systems for Chemistryand Life Sciences. San Antonio, Tex. (pages 1700-1703).

In embodiments, the present disclosure provides a substrate, an array, agrid, a microfluidic device, and the like, that includes an array ofmicrowells. In one embodiment, all of the microwells have essentiallythe same volume. This volume can be about 1 femtoliters, about 2, about4, about 6, about 8, about 10, about 20, about 40, about 60, about 80,about 100, about 200, about 400, about 600 about 800, or about 1,000femtoliters.

Moreover, the volume can take the form of a range between any of theabove two adjacent values, such as, the range of about 40 femtoliters toabout 60 femtoliters. Also, the volume can take the form of a rangebetween any of the above two values that are not immediately adjacent toeach other in the above list.

Furthermore, the volume can be about 1 picoliters, about 2, about 4,about 6, about 8, about 10, about 20, about 40, about 60, about 80,about 100, about 200, about 400, about 600 about 800, or about 1,000,about 2,000, about 5,000, about 10,000, about 20,000, about 50,000,about 100,000, about 200,000, about 500,000, or about 1,000,000picoliters. Also, the volume can take the form of a range between any ofthe above two values that are not immediately adjacent to each other inthe above list.

In exclusionary embodiments, the present disclosure can exclude anysubstrate comprising microwells, or any array comprising microwells,where the volume of each microwell is definable by one of the abovevalues, or is definable by a range of any of the above two values thatare adjacent to each other, or is definable by a range of any of theabove two values that are not adjacent to each other in the list.

Spherical Plug (Also Known as Capping Beads) on Microwells.

The present disclosure provides a spherical plug, or alternatively, aporous spherical plug, for each and every well, or substantially everywell of a picowell array. A goal of the plug is to keep drugs, drugcandidates, cellular contents, and metabolites, inside of the well. Theplug also helps isolate the contents of picowells from each other. Thespherical plug may need not be perfectly spherical, as long as the goalof covering the top (or opening, or mouth), of the picowell may besatisfied. The well can have a top diameter and a bottom diameter.Diameter of spherical plug, prior to capping a well, is about 10micrometers, about 30, about 35, about 40, about 45, about 50, about 55,about 70, about 90, about 120 or about 200 micrometers. The plugs may beadded to cover the picowells by simply flowing them over the picowellarray. Centrifugation, pressure, agitation or other methods may be usedto jam the beads to the tops (or mouths or openings) of the picowells toensure tight sealing. In some embodiments, solvents may be used tomodify the swelling and/or size of the capping beads. In someembodiments, the capping beads may be loaded in a solvent that rendersthe beads shrunken, and once replaced by assay buffer, or a differentsolvent, the capping beads are restored to their original sizes, orswell, thereby sealing the picowells tightly. In some embodimentstemperature may be used to swell or shrink the capping beads to obtainbetter seals at the mouths of picowells. Where needed, capping beads maybe held in place, and prevented from falling further into the picowell,by one of the steps in a stepped picowell array.

The capping beads may be the same type of beads that carry the compoundsof this disclosure, or may be beads of a different type. In someembodiments, the capping beads may actually be the compound bearingbeads themselves. The capping beads may serve as passive caps,preventing or slowing diffusion of molecules out of the picowells, orthe beads may be active beads, where functional moieties attached to thecapping beads may be used to capture reagents from the picowells. Insome embodiments, porous capping beads may passively trap metabolitesreleased from cell-based assays performed inside picowells. In someembodiments, capping beads may non-specifically capture cellularmaterials such as lipid, proteins, carbohydrates and nucleic acids. Insome embodiments, the capping beads may be functionalized withantibodies to specifically capture proteins released from healthy,diseased, lysed or fixed cells. In some embodiments, the capping beadsmay be functionalized with DNA or RNA oligonucleotides that specificallycapture cellular nucleic acids. In some embodiments, the DNA or RNAfunctionized capping beads may be used to capture microRNA released fromcells within the capped picowells. In some embodiments picowells containtwo beads, a compound containing bead inside the picowell, and a cappingbead covering the mouth of the picowells. In some embodiments, thecapping beads are also the compound-bearing beads. In some embodiments,the capping beads capture materials released from the compound beads. Insome embodiments, the capping beads capture a sampling of the compoundsreleased from compound-beads. In some embodiments, the capping beadscapture DNA barcodes released from the compound-beads. In someembodiments, the capping beads capture different types of analytesreleased from within the picowells they cap.

Relative Hardness of Cap and of Picowell.

A preferred equipment is a microwell plate, where each microwellincludes, in its bottom surface, many thousands of picowells. Ability ofa cap to seat properly or to seal each picowell can be a function of thehardness of the plastic that makes up the picowell's aperture and thepicowell's inner walls, relative to the hardness of the cap.

Hardness of a plastic can be defined in terms of a “durometer” value.Hardness is defined and tested as a material's resistance toindentation. The hardness of the spherical plug, and the hardness of thewall of the microwell can be defined in terms of its “durometer.” Thehardness can be, for example, about 45, about 50, about 55, about 60,about 65, about 70, about 75, about 80, about 85, about 90, about 95, orabout 100. In attributing any of these durometer values to a plasticsubstance or other substance, one must also state which scale is used.For example, the scale can be ASTM D2240 type A scale, which is used forsofter materials, or the ASTM D2240 type D scale, which is used forharder materials (see, Silicon Design Manual, 6^(th) ed., AlbrightTechnologies, Inc., Leominster, Mass.).

Shapes of Picowells.

In some embodiments, the picowells may be cylindrical picowells wherethe diameter of the cylinder is roughly similar at the top and thebottom of the picowell. In some embodiments, the picowells may have aslight taper, with the top of the picowells slightly larger than thebottom of the picowells. In some embodiments, the picowells may beconical picowells, with angles off normal anywhere between 1 degree to30 degrees. In some embodiments, the picowells are stepped picowells,where the picowells have discontinuous steps from the top diameter tothe bottom diameter (as opposed to conical picowells whose diameterchange smoothly from the top to the bottom). In some embodiments, thestepped picowells have a broad cylinder near the opening of the picowelland a narrower cylinder near the bottom of the picowells. In someembodiments, the stepped picowells may have multiple discontinuous stepsfrom the top to the bottom. In some embodiments of multi-steppedpicowells, the diameter at every rung may be larger than the diameter ofthe rung below it. In some embodiments a small bead may be deposited atthe bottom of the stepped picowell, and a capping bead may be depositedat the topmost opening of the stepped-picowell. In some embodimentspicowells may contain more than 2 beads.

Methods to Make Stepped-Picowells.

FIG. 29 disclosed stepped picowell. The embodiment shown has threecompartments and two steps. Top compartment is widest and is configuredfor accepting cap where most of the top compartment is occupied by thecap in the situation where the picowell is capped. Middle compartment isconfigured for being occupied mainly by, or solely by, reagents.Reagents can include buffer, enzyme substrates, one or more salts, and apreservative or stabilizer such as dithiothreitol, RNAse inhibitor,glycerol, or DMSO. Lowest compartment is configured for being occupiedby bead, that is, a bead with coupled both a DNA library and withreleasable compounds. In addition to bearing DNA barcode and releasablecompounds, the same bead can also bear a “response capture element.”Capping beads may be held in place, and prevented from falling furtherinto the picowell, by one of the steps in a stepped picowell. In FIG.29, structure 1 is cap., structure 2 is bead., and structure 3 is topregion, which is situated immediately above first step. Structure 4 ismiddle region, which can be used for placing assay reagents. Middleregion is immediately above second step. Assay reagents in middle regioncan diffuse into lowest region. Structure 5 is lowest region, which canbe used for placing a bead and for placing one or more cells.

Regarding the space of the lowest compartment that is taken up by thebead (assuming that only one bead is present in picowell), the diameterof the bead can be about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, or about 98%, of thediameter of the lowest compartment (assuming that the picowell is acircular well). If picowell is not a circular well, the above values canrefer to widest dimension of the well. In exclusionary embodiments, thepresent disclosure can exclude any system or bead that does not meet anyof the above parameters.

Further regarding space taken up by the bead (assuming that only onebead is present in picowell), about 50% of bead is in lowest compartmentand about 50% of same bead is in middle compartment, where theseparameters can also be: about 55% lowest and about 65% middle; about 60%lowest and about 40% middle; about 65% lowest and about 45% middle;about 70% lowest and about 30% middle, about 75% lowest and about 25%middle, about 80% lowest and about 20% middle, about 85% lowest andabout 15% middle, about 90% lowest and about 10% middle, about 95%lowest and about 5% middle, and about 100% lowest. For making thesecalculations the space taken up by bead assumes (hypothetically) thatthe bead is not porous. In exclusionary embodiments, the presentdisclosure can exclude any system or bead that does not meet any of theabove parameters.

As with conical and cylindrical picowell, using a molding system is onepreferred embodiment to create stepped picowells. For this purpose, amold containing arrays of multilayered pillars is desired, whereuponstamping into a thermoplastic or other curable polymer substrate, animpression of stepped picowells may be formed. A layered pillar arraywith multiple steps, each step of a different diameter (smaller as itgoes up) may be formed by a multilayer lithography process. Briefly, afirst layer of photoresist is exposed, via a first mask, to crosslinkthe first layer of the micropillar array. A second layer of photoresistmay be deposited directly on the (previously exposed) first layer, and asecond photomask may be used to crosslink a second pattern in the secondphotoresist later, and so on. At the end of the multiple-layerpatterning, the stack of resist may be developed to wash away theuncrosslinked regions, leaving an array of multilayered pillars.Detailed protocols for creating multilayered pillar arrays may be foundin Francisco Perdigones et al., (Jan. 8, 2011). Microsystem Technologiesfor Biomedical Applications, Biomedical Engineering, Trends inElectronics Anthony N. Laskovski, IntechOpen. Once an array ofmultilayered pillars array is created, standard processes may be used toimprint stepped picowell arrays using the mold.

Removing the Capping Beads.

In many embodiments it is advantageous to sample the capping beads tostudy reactions, analytes or cellular response to the chemicalperturbations within picowells. In some embodiments, the capping beadsmay be dislodged from the mouths of the picowells by inverting thepicowell array and using mechanical agitation. In some embodimentssolvents may be used to shrink the picowells, rendering them easier todislodge from the mouths of picowells. In some embodiments, liquids ofhigher density than the capping beads may be added on top of thepicowell array, causing the capping beads to raise by buoyancy and floatatop the high-density medium.

In some embodiments, the capping beads may be crosslinked to each other,converting the capping beads to a capping sheet that can be peeled offthe top of the picowell array. In some embodiments, a crosslinking gelmay be poured over the capped picowells, where the crosslinking gelcrosslinks to the capping beads, and to themselves, causing the cappingbeads to be embedded into a crosslinked sheet that can be peeled off.

Preserving Relative Locations of Picowells, in the Form of thePeeled-Off Layer.

It should be appreciated that in such embodiments as when the cappingbeads are enmeshed into a gel layer that can be peeled off, the relativelocations of capping beads with respect to each other and with respectto the picowells are preserved in the peeled-off layer. This allowsdirect connection between picowells, assays in picowells, the beads inthe picowells, and any materials captured in the capping beads.

In some embodiments, fiduial markers may be used to orient the relativefeatures of the picowell arrays to the capping beads in thepeeled-off-layer.

Fiducial Markers to Enable Registration and Alignment of Picowells.

Arranging picowells in irregular arrays allows easy identification ofshifts and drifts during imaging of the picowell arrays. In someembodiments, the picowells are arranged in an irregular order tofacilitate detection of optical and mechanical drifts during imaging. Insome embodiments, the picowell arrays contain fiducial markers to helpidentify shifts and drifts during imaging. In some embodiments, thefiducial markers are easily identifiable shapes, patterns or featuresthat are interspersed between the picowells of the picowell array. Insome embodiments, a small number of picowells may themselves be arrangedin an easily identifiable pattern to allow easy registration in case ofoptical or mechanical drifts during imaging. In some embodiments,external marker, such as fluorescent beads, may be drizzled on thepicowell array to provide fiducial patterns.

Cap-Free Mat Embodiments.

Cap-free mat embodiment, at least in some forms or examples, can takethe form of a “capless film.” Instead of sealing openings at the top ofpicowells, for example, for preventing evaporation of any cell culturemedium or enzyme assay medium that may be in the picowell, sealing canbe accomplished by way of a mat. Preferably, the mat is sized to coverall of the picowells in a given picowell array. Alternatively, the matcan be sized to cover a predetermined fraction of the picowells in thearray. The mat can be secured to the top of the picowell plate, coveringpicowells and also covering the generally planar top surface of thepicowell plate that resides in between the picowells. Secure contact canbe achieve by one or more of: (i) Maintaining constant pressure, forexample, by a hard rubber platen that sits on top of and serves as aweight on top of the mat; (ii) Using a mat that is connected to aweight, such as hard rubber platen; (iii) A reversible chemicaladhesive, that can be applied to the entire mat (in the situation wherethe mat is not be an absorbent mat). Where the mat is to be an absorbentmat, the mat contains circular absorbent pads that are surrounded by thereversible chemical adhesive. Here, the mat is contacted with thepicowell array and aligned so that the circular absorbent pads coveronly the openings of each picowell, and do not “spill out” over theopening to contact the planar surface of the picowell plate.

Membranes for use as mat for contacting substantially planar surface ofpicowell plate, and for use in capless-sealing of picowells, areavailable. Flat sheet membranes, such as Dow Film Tex, GE Osmonics,Microdyn Nadir, Toray, TriSep, Synder, Novamem, Evonik, and Aquaporinflatt sheet membreans are available from Sterlitech Corp, Kent, Wash.These include membranes made of polyamide-TFC, cellulose acetate,polyamide-urea-TFC, cellulose acetate blend, polypiperazine-amide-TFC,PES, composite polyamide-TFC, PES, PAN, PVDF, PSUH, RC, PESH, polyetherether ketone, polyimide, and so on. Pore size in terms of molecularweight cutoffs include, 150 Da, 200 Da, 300 Da, 500 Da, 900 Da, 600 Da,1,000 Da, 2,000 Da, 3,000 Da, 5,000 Da, 10,000 Da, 50,000 Da, 20,000 Da,30,000 Da 70,000 Da, 100,000 Da, 200,000 Da, 300,000 Da, 400,000 Da,500,000 Da, 800,000 Da, 3500 Da, 0.005 micrometers, 0.030 micrometers,0.05 micrometers, 0.10 micrometers, 0.20 micrometers, and so on.Regarding the system, compositions, reagents, and methods of the presentdisclosure, these cutoff values can allow selective collection ofcertain classes of compounds with exclusion of other classes ofcompounds. For example, some of the above membranes can allow smallmolecule metabolites to pass through and be absorbed by an absorbablemat, while excluding proteins and other macromolecules. Flat sheetmembranes that are impermeable to all molecules, including water, metalions, salts, metabolites, proteins, and nucleic acids, are alsoavailable for use in the systems, compositions, and methods of thepresent disclosure.

Reversible adhesion can be mediated by “molecular velcro,” for example,metalloporphyrin containing polymers with pyridine-containing polymers(Sievers, Namyslo, Lederle, Huber (2018) eXPRESS Polymer Letters.12:556-568). Other molecular velcro adhesives involve,L-3,4-dihydroxyphenyl alanine, complementary strands of ssDNA (one typeof ssDNA covalently attached to flat upper surface of picowell plate,and other type of ssDNA covalently attached to mat), copolymerscontaining catechol side chains, and so on (see, Sievers, et al, supra).Also, reversible adhesion can be mediated by a gallium adhesive, wheredegree of adhesion can be controlled by slight changes in temperature(Metin Sitti (May 18, 2016) Switch and Stick. The chemical elementgallium could be used as a new reversible adhesive that allows itsadhesive effect to be switched on and off with ease.Max-planck-Gesellschaft). Yet another reversible adhesive is availablefrom DSM-Niaga Technology, Zwoll, The Netherlands.

Absorbent Substances (Non-Specific Absorbents; Specific Absorbents).

Absorbent substances, which can be incorporated into a mat to provideabsorbent characteristics include “molecule sieve” beads, such asSepharose®, Sephadex®, Agarose®, as well as ion exchange beads made ofDEAE cellulose, carboxymethylcellulose, phosphocellulose, or anycombination of the above, all combined into one absorbent mat. Absorbentligands include those that are used in high pressure liquidchromatography (HPLC) (see, BioRad catalog, Hercules, Calif.). Specificabsorbents include response-capture elements, such as poly(dT), whichcan capture mRNA by way of hybridizing with polyA tail. Also, responsecapture elements include exon-targeting RNA probs, antibodies, andaptamers. Each or any combination of these can be covalently attached tomat, to create an absorbent mat, where contacting absorbent mat to topsurface of picowell enables capture of aqueous assay medium or aqueouscell culture medium that might be inside picowells.

(IX) Depositing Beads into Picowells

Plates with picowells can take the form of a 96-well plate where each ofthese 96 wells contains many thousands of picowells. Also, plates withpicowells can take the form of a 24-well plate, where each of these 24wells contains many thousands of picowells. For the 96-well plate, eachwell can be filled using 0.1-0.2 mL of a suspension of beads in water orin an aqueous solution. For the 24-well plate, each well can be filledusing about 0.5 mL of a suspension of beads in water or in an aqueoussolution. Suspension can be added using an ordinary pipet with adisposable tip. The number of beads that are in the suspension can bethat resulting in about one third of the picowells containing only onebead, about one third of the picowells containing two beads, and aboutone third of the beads containing either no beads or more than twobeads. Also, the number of beads in the suspension can be that resultingin the situation where, of the wells that do contain one or more beads,at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, orat least 98% of these wells contain only one bead.

After the beads have settled, any excess liquid can be removed bytouching a pipet tip to the wall of each well of the 96 well plate, orby touching a pipet tip to the wall of each well of the 24 well plate,and drawing off the excess liquid.

Regarding assay reagents, where the picowells are to be used forcarrying out reactions, for example, DNA sequencing, biochemical assays,or assays of cultured cells, assay reagents can be added to thepicowells that already contain settled beads. Adding the assay reagentsis with a pipet, as described above for initial addition of the beadsuspensions. After the assay reagents have equilibrated with thesolution that is already in each picowell, any excess solution that isin each of the 96 wells of the 96-well plate, or any excess solutionthat is in each of the 24 wells of the 24-well plate, can drawn off witha pipet tip that touches the wall of each of the 96 wells of the 96-wellplate, or that touches the wall of each of the 24 wells of the 24-wellplate.

Flow-Cell Embodiment of Picowell Array.

Picowell array may be part of a flow-cell, where a fluidic chamber withan inlet and an outlet are mounted on top of the picowell array. In suchembodiments, beads of this disclosure, cells, and other assay materialsmay be flowed in from the inlet and out through the outlet. Gravity orcentrifugal force may be used to lodge the beads into the picowells asthey are flowed through the flowcell.

(X) Sequencing Bead-Bound Nucleic Acids in Picowells

Bead-bound nucleic acids can be sequenced while still attached to beads.Alternatively, or in addition, bead-bound nucleic acids can be sequencedfollowing cleavage of the DNA barcode from the bead.

Cleaving the DNA Barcode from the Bead Before Sequencing.

In some embodiments, the present disclosure can encompass a method wherebead-bound DNA barcode is cleaved from the bead, thereby releasing theDNA barcode in a soluble form, prior to amplification, or prior tosequencing, or prior to any type of sequence identification techniquesuch as hybridizing with a nucleic acid probe.

Exclusionary Embodiments

In embodiments, the present disclosure can exclude any method,associated reagents, system, compositions, or beads, where a bead-boundDNA barcode is cleaved prior to amplification, or prior to sequencing,or prior to any type of sequence identification technique such ashybridizing with a nucleic acid probe. Also, the present disclosure canexclude any method where a polynucleotide comprising a DNA barcode iscleaved, or where a nucleic acid comprising only part of a DNA barcodeis cleaved, prior to amplification, prior to sequencing, or prior to anytype of sequence identification technique such as hybridizing with anucleic acid probe.

Polymerase Chain Reaction (PCR); Quantitative PCR (qPCR).

The PCR method, as well as the qPCR method, depend on the 3-step methodinvolving: (1) Denaturing the DNA template at a high temperature,annealing primers at a reduced temperature, and finally extending theprimer by way of DNA synthesis, as catalyzed by DNA polymerase (Gadkarand Filion (2014) Curr. Issues Mol. Biol. 16:1-6). qPCR is also called,“real time PCR” (Kralik and Ricchi (2017) Frontiers Microbiology. 8 (9pages).

Recent modifications or improvements in the PCR method and qPCR methodinclude, using helicase-dependent (HDA) amplification, using an internalamplification control, using locked nucleic acids (LNA), and usingadditives that bind to inhibitors (Gadkar and Filion (2014) Curr. IssuesMol. Biol. 16:1-6). Locked nucleic acids provide the advantage ofrecognizing and binding its target with extreme precision.

qPCR allows the simultaneous amplification and quantification of atargeted DNA molecule. The qPCR method compares the number ofamplification cycles required for the response curvecs to reach aparticular fluorescence threshold (Pabinger, Rodiger, Kriegner (2014)Biomolecular Detection Quantification. 1:23-33). Refsland et al providea concise account of apparently typical conditions for conducting qPCR(Refsland, Stenglein, Harris (2010) Nucleic Acids Res. 38:4274-4284).

Guidance is available for designing and validating PCR primers, and onvariables such annealing temperature (Ta), melting temperature (Tm),temperature of elongation step, type of buffer (Bustin and Huggett(2017) Biomolecular Detection Quantification. 14:19-28).

Rolling Circle Amplification (RCA).

DNA can be amplified while attached to a bead. DNA in amplified form iseasier to sequence that non-amplified DNA. In the rolling circleamplification method, DNA tags (the DNA barcode) is made singlestranded. Once single stranded, a splint oligo is added to bridge theends of the tag DNA, and this is followed by extension and ligation ofthe splint oligo. Using DNA polymerase (minus 5′→3′exonuclease activity)ensures a ligatable junction after the DNA catalyzes extension of thesplint oligo. The circularized DNA can then be subjected to rollingcircle amplification by adding a strand-displacing DNA polymerase, suchas phi29 DNA polymerase. The ability to perform rolling circleamplification (RCA) on the DNA barcode tag permits the use of synthesischemistries that may be damaging to DNA, as any surviving DNA moleculescan be thermally amplified to sufficient quantities to be easilysequenced. DNA can be made single-stranded by exonuclease digestion,nicking, and melting at high temperature, or by treating with sodiumhydroxide.

Further details of rolling circle amplification (RCA) are revealed bythe following steps that can be used for conducting RCA.

Step One:

Start with bead-bound ssDNA. If the bead-bound DNA is initially in adouble stranded from (dsDNA), the strand that is not to be used for RCAcan be prepared so that a residue of thymine (T) is replaced, at or veryclose to the bead-attachment terminus, with a residue of uracil (U). Ifthe dsDNA is prepared in this way, uracil-N glycosidase can be used tocleave the uracil residue, thereby leaving an unstable sugar phosphate(as part of the DNA backbone), where this unstable location can becleaved by nuclease-treatment (Ostrander et al (1992) Proc. Natl. Acad.Sci. 89:3419-3423).

Step Two:

Add a “splint oligo” to the bead-bound ssDNA. The splint oligo isdesigned so that it hybridizes to about 10-20 base pairs at the end (the5′-end) of the ssDNA that is covalently coupled to the bead, and so thatit also hybridizes to about 10-20 base pairs at the free end (the3′-end) of the bead-bound ssDNA. The splint oligo does not need to bringthe bead-bound end of the ssDNA in close proximity to the free end ofthe bead-bound ssDNA. All that is needed is for the far ends of thebead-bound ssDNA sequence be tethered together, in order to form a hugeloop.

Step Three:

Add sulfolobus DNA polymerase IV, so that this polymerase uses the hugeloop of ssDNA as a template, for creating a complementary huge loop thatis covalently attached at one end to the splint oligo.

Step Four:

Use DNA ligase to covalently close the complementary huge loop, wherethe result is circular ssDNA. It is this closed circle of ssDNA thatdoes the “rolling,” during RCA.

Step Five:

Add DNA polymerase that has a strand displacement activity, and adddNTPs. The added DNA polymerase covalently attaches dNTPs to thebead-bound ssDNA, and the distal terminus of the bead-bound ssDNA isextended to create a complementary copy of what is on the “rollingcircle,” and then further extended to create yet another complementarycopy of what is on the “rolling circle,” and even more extended tocreate still another complementary copy of what is on the “rollingcircle.” During this process of potentially infinite amplification,continued activity of DNA polymerase is made possible by the stranddisplacement activity of the DNA polymerase.

Optionally, the method of the present disclosure includes real-timemonitoring of rolling circle amplification (RCA) by way of fluorescentmolecular beacons (Nilsson, Gullberg, Raap (2002) Nucleic Acids Res.30:e66 (7 pages)). Reagents for RCA are available from Sigma-Aldrich(St. Louis, Mo.), Sygnis TruePrime Technology (TruePrime® RCA kit),Heidelberg, Germany, and GE Healthcare (TempliPhi 500® amplificationkit). Fluorophores and quenchers are available from ThermoFisherScientific (Carlsbad, Calif.), Molecular Probes (Eugene, Oreg.), CaymanChemical (Ann Arbor, Mich.), and Sigma-Aldrich (St. Louis, Mo.).

Step Six.

Use the ssDNA that was amplified by RCA as a template for PCRamplification, where primers are added, where thermostable DNApolymerase is added, and where the PCR products are subsequentlysequenced by Next Generation Sequencing.

In one aspect of the present disclosure, the RCA-amplified ssDNA iscleaved from the bead prior to PCR amplification that makes PCRproducts. In another aspect of the present disclosure, the PCRamplification that makes PCR products can be made without cleaving theRCA-amplified ssDNA from the bead.

As described by Baner et al, “Through the RCA reaction, a strand can begenerated that represents many tandem copies of the complement to thecircularized molecule” (Baner, Nilsson, Landegren (1998) Nucleic AcidsRes. 26:5073-5078). Bacillus subtilis phase phi29 DNA polymerase is asuitable enzyme, because of its strand displacement activity and highprocessivity. RCA is similarly characterized by Li et al as, “In RCA, acircular template is amplified isothermally by a DNA polymerase phi29with . . . strand displacement properties. The long single-stranded DNAproducts contain thousands of sequence repeats: (Li and Zhong (2007)Anal. Chem. 79:9030-9038).

Sequencing of DNA barcodes of the present disclosure can be, withoutimplying any limitation, with methods of Vander Horn U.S. Pat. No.8,632,975, which is incorporated herein by reference in its entirety.Also, the DNA barcodes of the present disclosure can be sequenced, forexample, by methods that use sequencing-by-synthesis, such as the Sangersequencing method, or by methods that use “Next Generation sequencing.”

Illumina Method for DNA Sequencing.

Illumina method for DNA sequencing is as follows. DNA can be fragmentedto a size range of 100-400 base pairs (bp) by sonication (Hughes,Magrini, Demeter (2014) PLoS Genet. 10:e1004462). In the Illuminamethod, DNA libraries are made, where fragments of DNA from a cell orfrom cells are modified by DNA adaptors (attached to termini of thefragments). The reaction product takes the form of a sandwich, where theDNA to be sequenced is in the center of the sandwich. The reactionproduct takes the form: (first adaptor)-(DNA to be sequenced)-(secondadaptor). The adaptor-DNA-adaptor complex is then associated with yetanother adaptor, where this other adaptor is covalently attached to asolid surface. The solid surface can be a flat plate. The solid surfacehas a lawn of many adaptors that stick out of the flat surface. Theadaptor has a DNA sequence that is complementary to one of the adaptorsthat is in the sandwich. Actually, the lawn contains two type ofadaptors, where one adaptor binds (hybridizes) to one of the adaptors inthe complex, and non-covalently tethers the complex to the plate. Thesemay be called the, “first lawn-bound adaptor” and the “second lawn-boundadaptor.” The first task of DNA polymerase, is to create a daughterstrand, using the tethered (but non-covalently bound) DNA as a templateand, when DNA polymerization occurs, the daughter strand is in a formthat is covalently attached to the “first lawn-bound adaptor.” Thiscovalent link was generated by the catalytic action of DNA polymerase.After the daughter strand is completely sythesized, the distal end (theend that sticks out into the medium) contains a DNA sequence that iscomplementary to the second adaptor in the above-named sandwich. ThisDNA sequence that is complementary, allows the distal end of the newlysynthesized daughter DNA to bend over and to hybridize to the “secondlawn-bound adaptor.” What has been described above, is how both adaptorsof the sandwich are used, and how both the “first lawn-bound adaptor”and the “second lawn-bound adaptor” are used.

A cycle of reactions is then performed many times, where the result is acluster of amplified versions of the original dsDNA. Actually, thecluster takes the form of covalently attached (tethered) ssDNAmolecules, where all of these ssDNA molecules correspond to only one ofthe strands of the original dsDNA (dsDNA isolated from a living cell ortissue). This cluster of tethered ssDNA molecules is called a “polony.”The generation of the polony is by a technique called, “bridgeamplification.” Finally, after bridge amplification and the creation ofpolonies, the reverse strands that are covalently attached to the solidsurface are cleaved from its tetherings, washed away, and discarded,leaving only the forward strands.

Information on the Illumina® method is available from Goodwin,McPherson, McCombie (2016) Nature Rev. Genetics. 17:333-351, Gierahn,Wadsworth, Hughes (2017) Nature Methods. 14:395-398, Shendure and Hanlee(2008) Nature Biotechnology. 26:1135-1145; Reuter, Spacek, Snyder (2015)Molecular Cell. 58:586-597; Illumina Sequencing by Synthesis (5 minutevideo on YouTube).

Sequencing by Oligonucleotide Ligation and Detection (SOLiD Sequencing).

SOLiD measures fluorescence intensities from dye-labeled molecules todetermine the sequence of DNA fragments. A library of DNA fragments isprepared from the sample to be sequenced and used to prepare clonal beadpopulations (with only one species of fragment on the surface of eachmagnetic bead). The fragments attached to the beads are given auniversal P1 adapter sequence attached so that the starting sequence ofevery fragment is both known and identical. PCR is conducted and theresulting PCR products that are attached to the beads are thencovalently bound to a slide.

Then, primers hybridize to the P1 adapter sequence within the librarytemplate. A set of four fluorescently labelled di-base probes competefor ligation to the sequencing primer. Specificity of the di-base probeis achieved by interrogating every 1st and 2nd base in each ligationreaction. Multiple cycles of ligation, detection and cleavage areperformed with the number of cycles determining the eventual readlength. Following a series of ligation cycles, the extension product isremoved and the template is reset with a primer complementary to the n−1position for a second round of ligation cycles (see, Wu et al (2010)Nature Methods. 7:336-337).

pH-Based DNA Sequencing.

pH-Based DNA sequencing is a system and method where, baseincorporations are determined by measuring hydrogen ions that aregenerated as byproducts of polymerase-catalyzed extension reactions. DNAtemplates each having a primer and polymerase operably bound are loadedinto reaction chambers or microwells, after which repeated cycles ofdeoxynucleoside triphosphate (dNTP) addition and washing are carriedout. The DNA template is templates are attached as clonal populations toa solid support. With each such incorporation a hydrogen ion isreleased, and collectively a population of templates releasing hydrogenions causing detectable changes to the local pH of the reaction chamber(see, Pourmand (2006) Proc. Nat'l. Acad. Sci.103:6466-6470). The presentdisclosure can exclude pH-based DNA sequencing.

Regarding the concatenated DNA barcode, the entire concatenated DNAbarcode can be sequenced in one run (where sequencing of the entireconcatenated DNA barcode requires only one sequencing primer).Alternatively, some or all of the DNA barcode modules that make up theconcatenated DNA barcode can be subjected to individual sequencing(where each of the individually-sequenced DNA barcode modules gets itsown sequencing primer). Regarding orthogonal DNA barcodes, each of theDNA barcode modules that make up the orthogonal DNA barcode needs itsown, dedicated sequencing primer, because of the fact that each DNAbarcode module is attached to its own site on the bead.

Exclusionary Embodiments

In embodiments, the present disclosure can exclude any system, device,combination of devices, and method, that involves microfluidics, aqueousdroplets that reside in an oil medium, and aqueous droplets that arecreated where a first channel containing aqueous reagents is joined witha second channel containing an oil to create aqueous droplets that movethrough an oil medium through a third channel that starts at the joiningarea. Microfluidics devices and reagents are described (see, e.g.,Brouzes, Medkova, Savenelli (2009) Proc. Natl. Acad. Sci.106:14195-14200; Guo, Rotem, Hayman (2012) Lab Chip. 12:2146-2155; Debs,Utharala, Balyasnikova (2012) Proc. Natl. Acad. Sci. 109:11570-11575;Sciambi and Abate (2015) Lab Chip. 15:47-51).

In other exclusionary embodiments, what can be excluded is any reagent,composition, nucleic acid, or bead, that comprises a “DNA headpiece” oran reagent, composition, nucleic acid, or bead, that is covalentlyattached to a “DNA headpiece.” MacConnell, Price, Paegel (2017) ACSCombinatorial Science. 19:181-192, provide an example of a DNAheadpiece, where beads are functionalized with azido DNA headpiecemoieties.

Additional Exclusionary Embodiments Relating to Sequencing Methods andSequencing Reagents.

In embodiments, the present disclosure can exclude reagents, systems, ormethods that do not involve use of a “reversible terminator” in DNAsequencing. Also, what can be excluded is any reagent, system, ormethod, that do not include methoxy blocking group. Moreover, what canbe excluded is any reagent, system, or method, that involves DNAsequencing, but where the DNA being sequenced is not covalently bound toa bead at the time at the time that information on the order ofpolynucleotides is being detected and collected. Furthermore, what canbe excluded is any reagent, system, or method that amplifies a DNAtemplate prior to conducting sequencing reactions, for example,amplification by PCR technique or by rolling circle technique. Inembodiments, what can be excluded is any method of barcoding, forexample, nucleic acid barcoding, that is concatenated (all informationon synthesis of a member of the chemical library residing on one singlenucleic acid). In another aspect, what can be exluced is any method ofbarcoding, for example, nucleic acid barcoding, that is orthogonal(information on synthesis of a given monomer of a chemical library beingdispersed on a plurality of attachment positions on the bead). In anexclusionary embodiment relating to DNA ligase, the present disclosurecan exclude any reagent, system, or method, that uses DNA ligase forconnecting modules of a nucleic acid barcode.

Fluorophores, Quenchers, and FRET-Based Assays.

The present disclosure provides fluorophores and quenchers for screeningmembers of a chemical library, or for characterizing an isolated memberof a chemical library. FRET is Forster resonance energy transfer.

Assays can be performed on bead-bound chemical libraries. Also, assayscan be performed on free chemical library members shortly after cleavagefrom a bead, that is, performed in the same microwell as the bead orperformed in the same vicinity of a hydrogel matrix as the bead.Moreover, assays can be performed on a soluble chemical library memberthat had never been attached to any bead, or that had been cleaved froma bead and then purified.

Fluorophores suitable as reagents of the present disclosure includeAlexa 350, Alexa 568, Alexa 594, Alexa 633, A647, Alexa 680,fluorescein, Pacific Blue, coumarin, Alexa 430, Alexa 488, Alexa 532,Alexa 546, Alexa 660, ATTO655, ATTO647n, Setau-665 (SETA Biochemicals,Urbana, Ill.), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, tetramethylrhodamine (TMR),Texas red, tetrachlorofluorescein (TET), hexachlorofluorescein (HEX),and Joe dye (4′-5′-dichloro-2′,7′-dimethoxy-6-carboxyfluorescein), SYBRgreen I (absorb 497 nm, emit 520 nm), 6-carboxyfluorescein (6-FAM)(absorbs 492 nm, emits 518 nm), 5-carboxyfluorescein (5-FAM) (absorbs492 nm, emits 518 nm), FITC, and rhodamine. Quenchers include TAMRAquencher, black hole quencher-1 (BHQ1), and black hole quencher-2(BHQ2), and DABCYL quencher. Please note, as disclosed elsewhere in thispatent document, that TAMRA can be a fluorophore and it can also be aquencher.

Guidance is available on reagents for FRET-based assays, where the FRETreagent includes a fluorophore and quencher (see, Johansson (2006)Choosing reporter-quencher pairs for efficient quenching. Methods Mol.Biol. 335:17-29). An example of a FRET-based assays including measuringthe activity of a signal peptidase (SpsB) with the substrate, “SceDpeptide.” The FRET-pair attached to the peptide was4-(4-dimethylaminophenylazo) 5-((2-aminoethyl)amino)-1-nepthalenesulfonic acid (see, Rao et al (2009) FEBS J.276:3222-3234). Another example comes from assays of HIV-1 protease,with the peptide substrate, KVSLNFPIL. The donor/acceptor FRET pair wasEDANS (donor) and DABCYL (acceptor). EDANS fluorescence can be quenchedby DABCYL by way of resonance energy transfer to the nonfluorescentDABCYL (see, Meng et al (2015) J. Biomolecular Screening. 20:606-615).Yet another example comes from assays of botulinum toxin. Activity ofSNAP-25 can be measured by using the substrate, BoNT-A. For FRET-basedassays, the substrate had an N-terminally linkedfluorescein-isothiocyanate (FITC) and the C-terminally linked quencherwas, 4-(4-dimethylaminophenyl) diazenylbenzoic acid (DABSYL). Thepeptide substrate corresponded to amino acids 190-201 of SNAP-25 (see,Rasooly and Do (2008) Appl. Environ. Microbiol. 74:4309-4313).

The present disclosure provides for reagents, compositions, and methodsfor screening a library of compounds in order to discover and identifyenzyme inhibitors, enzyme activators, and to discover compounds that canenhance the rate of in vivo degradation of a given protein. Thesereagents, compositions, and methods can use FRET-based assays and,alternatively, they can use assays other than FRET-based assays.

Molecular beacons are described (see, Baruch, Jefferey, Bogyo (2004)Trends Cell Biology. 14:29-35). A molecular beacon is a reagent where afluorophore is bound, by way of a linker, to a quencher. The linker maybe cleavable by a nuclease, and thus measure nuclease activity. Thepresent disclosure provides for methods to screen chemical libraries foridentifying nuclease inhibitors and, alternatively, for identifyingnuclease activators. Feng et al have described the use of molecularbeacons and use of FRET-based assays for measuring activity of variousnucleases (Feng, Duan, Liu (2009) Angew Chem. Int. Ed. Engl.48:5316-5321). Feng et al, showed use of FRET-based assays for measuringactivity of various restriction enzymes.

(XI) Releasing Bead-Bound Compounds

Cleavable Linkers.

What is provided is linkers that are not cleavable. Also, what isprovided are cleavable linkers (see, Holmes and Jones ((1995) J. Org.Chem. 60:2318-2319; Whitehouse et al (1997) Tetrahedron Lett.38:7851-7852, and Yoo and Greenberg ((1995) J. Org. Chem. 60:3358-3364,as cited by Gordon et al (1999) J. Chem. Technology Biotechnology.74:835-851). Cleavable linkers also include an acyl sulphonamide linkersthat reside alkaline hydrolysis, as well as activated N-alkylderivatives which are cleaved under mild conditions, and also tracelesslinkers based on aryl-silicon bonds, and traceless linkers based onsilyl ether linkages (described on page 839 and 842 of Gordon et al(1999) J. Chemical Technology Biotechnology. 74:835-851). Moreover, whatis provided is a linker based on tartaric acid which, upon cleavage,generates a C-terminal aldehyde, where cleavage is by periodateoxidation (see, Paulick et al (2006) J. Comb. Chem. 8:417-426).

FIGS. 3A-3I disclose various cleavable linkers that are suitable for thecompositions and methods of the present disclosure. FIGS. 3A-3I arereproduced from Table 1 of: Yinliang Yang (2014) Design of CleavableLinkers and Applications in Chemical Proteomics. Technische UniversitatMunchen Lehrstuhl fur Chemie der Biopolymere. From FIGS. 3A-3I,cleavable linkers that are preferred for the present disclosure arelinkers A, C, D, E, F, G, and I. Linker E was used in the experimentalresults disclosed herein. Cleavage conditions for these are DTT (linkerA), Na₂SO₄ (linker C), Na₂SO₄ (linker D), UV light (linker E), UV light(linker F), UV light (linker G), and TEV protease (linker I). Theseparticular cleavage conditions are gentle and are not expected to damagethe bead, to damage the bead-bound compound, or to damage any chemicallibrary member (the unit) of the bead-bound compound.

Chemically Cleavable Linkers that are Compatible with Click-Chemistry.

Qian et al (2013) describes a number of cleavable linkers that arecompatible with click-chemistry (Qian, Martell, Pace (2013) ChemBioChem.14:1410-1414). These include linkers with an azo bond, where the azobond is cleavable with dithionite. This linker has the followingstructure: R₁-benzene1-N═N-benzene₂-R₂. The first benzene ring has ahydroxy group para to R₁, and the second benzene ring has a carbonylgroup that links to R2, where this carbonyl group is para to the azomoiety.

Photolabile Cleavable Linkers.

The present disclosure encompasses photocleavable linkers that have ano-nitrobenzyl group. This group can be cleaved by irradiation at 330-370nm (see, Saran and Burke (2007) Bioconjugate Chem. 18:275-279;Mikkelsen, Grier, Mortensen (2018) ACS Combinatorial Science.DOI:10.1021). A linker with a shorter photolysis time than o-nitrobenzyllinker is 2-(2-nitrophenyl)-propyloxycarbonyl (NPPOC) linker. Avariation of o-nitrobenzyl linker is o-nitrobenzylamino linker. Whenattached to a peptide chain, and when subsequently cleaved, this linkerreleases an amide. Linker with an o-nitroveratryl group are available,and these have shorter photolysis time and greater release yields thanunsubstituted o-nitrobenzyl linkers. Also available are phenacyllinkers, benzoin linkers, and pivaloyl linkers (see, Mikkelsen et al(2018) ACS Combinatorial Science. DOI:10.1021).

Linkers with photocleavable ether bonds are available. Thisphotocleavable linker can be used where the linker is attached to a beadand where the cleavable group is an “R group,” and after cleavage, thereleased group takes the form of ROH (see, Glatthar and Giese (2000)Organic Letters. 2:2315-2317). Also available are linkers withphotocleavable ester bonds (see, Rich et al (1975) 97:1575; Renil andPillai (1994) Tetrahedron Lett. 35:3809-3812; Holmes (1997) J. Org.Chem. 62:2370-2380, as cited by Glatthar and Giese, supra). Ether bondsin linkers can be cleaved by acid, base, oxidation, reduction, andfluoride sensitive silyl-oxygen bond cleavage, and photolysis (Glattharand Giese, supra).

Another photocleavable linker, which has been used to link a peptide(R₁) and a nucleic acid (R₂), is as follows. R₁ is connected directly tothe methylene moiety of a benzyl group. Para to the methylene group is aring-attached nitro group. Meta to the methylene moiety is aring-attached ethyl group. The 1-carbon of the ethyl group bears aphosphate. To an oxygen atom of this phosphate is attached the R₂ group(Olejnik et al (1999) Nucleic Acids Res. 27:4626-4631).

Akerblom et al, discloses photolabile linkers of the alpha-methyl2-nitrobenzyl type, containing amino, hydroxyl, bromo, and methylaminogroups, and also 4-nitrophenoxycarbonyl activated hydroxyl and aminogroups (see, Akerblom and Nyren (1997) Molecular Diversity. 3:137-148).Cathepsin B can cleava a linker with the target sequence,“valine-citrulline” (Dal Corso, Cazzamalli, Neri (2017) BioconjugateChemistry. 28:1826-1833).

Enzyme-Cleavable Linkers.

Linkers that are cleavable by enzymes, such as proteases, are available(see, Leriche, Chisholm, Wagner (2012) Bioorganic Medicinal Chem.20:571-582). The hydroxymethylphenoxy linker can be cleaved withchymotrypsin (Maltman, Bejugam, Flitsch (2005) Organic BiomolecularChem. 3:2505-2507). Linkers that are cleavable with tobacco etch virusprotease are available (see, Weerapana, Speers, Cravatt (2007) NatureProtocols. 2:1414-1425; Dieterich, Link, Graumann (2006) Proc. Nat'l.Acad. Sci. 103:9482-9487). The linker sequences LVPRG and LVPRGS can becleaved by thrombin (Jenny, Mann, Lundblad (2003) Protein ExpressionPurification. 31:1-11). Plasmin-cleavable linkers are available (Devy,Blacher, Noel (2004) FASEB J. 18:565-567).

Bead-Bound Release-Monitor.

The present disclosure provides a novel and unique release-monitor thatis capable of assessing release of bead-bound compounds. Therelease-monitor takes the form of a bead-bound complex of fluorophoreand quencher, where the fluorophore is connected to the bead by way of acleavable linker. Preferably, the cleavable linker is a photocleavablelinker. Preferably, the bead-bound release-monitor is situated in adedicated picowell, where that picowell does not contain any other typeof bead. With severing of the photocleavable linker, the fluorophore isreleased from the bead, diffuses into the medium in the picowell,achieves some distance from the bead-bound quencher, where the result isan increase in fluorescence that is proportional to the amount ofrelease. The increase in fluorescence allows the calculation of theconcentration of the free fluorophore that is in the picowell and, moreimportantly, allows calculation of the amount of chemical compounds thatare released from other beads that are situated in other wells.

To summarize, the bead-bound release-monitor is situated in its owndedicated well, where other wells contained bead-bound compounds thatare drug candidates.

FIG. 8 discloses a simplified version of a preferred and non-limitingexample of a bead-bound release-monitor. The release-monitor takes theform of a quencher that is held in the vicinity of a fluorophore,resulting in quenching of the fluorophore. In embodiments, quenching isat least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%,at least 99.95%, and so on. In a picowell, one bead is dedicated tobeing a release-monitor, while another bead or beads are used forattaching a compound and for attaching DNA library. Exposure of all ofthe beads in a picowell to UV light result in simultaneous cleavage offluorophore and of the compound. QSY7 is a preferred quencher. Thestructure and CAS number for QSY7 is as follows (see below):

CAS name/number: Xanthylium, 9-[2-[[4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-1-piperidinyl]sulfonyl]phenyl]-3,6-bis(methylphenylamino)-,chloride 304014-12-8

The increase in fluorescence that results from separation of thefluorophore from the quencher can be used to infer the concentration inthe picowell of the simultaneously released compound. Also, the increasein fluorescence that results from separation of the fluorophore from thequencher can be used to infer the number of molecules (molecules takingthe form of the compound that was formerly a bead-bound compound) thatreside in free form in the picowell. In a more preferred embodiment, therelease-monitor comprises a quencher and a fluorophore, where cleavageresults in the release of the fluorophore (and not release of thequencher). This embodiment provides lower background noise than thefollowing less preferred embodiment. In a less preferred embodiment,cleavage results in the release of the quencher, where the read-outtakes the form of the increase in fluorescence from bead-boundfluorophore.

The release-monitor provides the user with a measure of theconcentration of the soluble compound, following UV-induced release ofthe compound from the bead. In a preferred embodiment, one type of beadis dedicated to being a release-monitor. By “dedicated,” what this meansis that this bead does not also contain bead-bound compound and does notalso contain bead-bound DNA library.

As a general proposition, just because a compound has been released froma bead by cleavage of a photosensitive linker, it should not be inferredthat the compound has become a soluble compound. First of all, pleasenote that just because a compound is considered to be “hydrophobic” oris considered to be “water-insoluble” does not mean that none of themolecules are freely moving in the solvent. For example, evencholesterol has a measurable solubility in water (see, Saad and Higuchi(1965) Water Solubility of Cholesterol. J. Pharmaceutical Sciences.54:1205-1206). Moreover, biochemical efficacy of a bead-boundwater-insoluble compound can be increased, by way of surfactants,detergents, additives such as DMSO, or carriers such as human serumalbumin. Thus, the release-monitor can be used to assess overallconcentration of compounds of limited water-solubility or of nowater-solubility, under the condition where the picowell contains one ofthe above agents or, alternatively, where the water-insoluble compoundis released in the vicinity of the plasma membrane of a living cell thatis cultured inside of the picowell.

FIG. 9 discloses a simplified version of a preferred embodiment ofbead-bound release-monitor, while FIG. 10 discloses a complete anddetailed structure of this preferred embodiment of bead-boundrelease-monitor.

FIGS. 30A-30F provide data demonstrating use of bead-release monitor,where bead is in a picowell. The bead-bound fluorophore, which is boundusing a light-cleavable linker, was TAMRA (excitation wavelength 530 nm;emission wavelength 570 nm). The figure shows time-course of release ofthe fluorophore from the bead. This shows operation of the bead-boundrelease monitor, acquisition of fluorescent data at t=0 seconds, t=1seconds, t=11 seconds, and t=71 seconds. FIGS. 30A-30F also includeinsets showing blowups of the smaller figures, for two of the foursmaller figures. FIGS. 30A-30F were obtained from incubation ofcathepsin-D, which is an aspartyl protease, with “Peptide Q-FluorSubstrate” and beads. Reagents were placed into wells at 4 degrees C.Ultraviolet light at 365 nm was used to cleave the fluorophore from thebead, thereby releasing the fluorophore and separating it from thequencher. A goal of this assay was to assess the time course of releasetaking place in a separate well, where the separate well contained adifferent type of bead. The different type of bead had the samelight-cleavable linker, but where this light-cleavable linker wasattached to pepstatin-A. Release of pepstatin-A can bind to and inhibitan aspartyl protease that is in the same assay medium. This setup withbead-bound peptstatin-A and the aspartyl protease can serve as apositive control.

UV exposed through 20× objective. Image was obtained with Gain=5;Exposure was 400 ms. Excite at TAMRA at 530 nm. TAMRA emits at 570 nm.

FIG. 35 discloses further details on enzymatic assays, where bead-boundpepstatin-A is released, and where the released pepstatin-A results inenzyme inhibition. 10 μm TentaGel beads displaying photocleavablePepstatin-A (positive control) and a covalent Cy5 label, were mixed with10 μm TentaGel beads displaying photocleavable Fmoc-Valine (negativecontrol) in PBST buffer. This bead population was introduced intopicowells, then buffer exchanged into a protease inhibition assay,including Cathepsin-D protease and Peptide Q-Fluor substrate (λ_(ex)=480nm, λ_(em)=525 nm). Wells were encapsulated by air, and entire slideexposed to UV (365 nm, 77 J/cm²), cleaving the photolabile linker,releasing the compound to reach approximately 13 μM. The flowcell wasincubated (30 min, 37° C.). Wells containing positive control beadsshould inhibit peptide proteolysis by Cathepsin-D, resulting in lowfluorescence signal. Wells containing negative control beads should notshow any Cathepsin-D inhibition, and should be similar in fluorescenceintensity to empty wells.

Terminology for Quencher and Fluorophore can Change, for a GivenChemical, Depending on What Other Chemicals Occur in the ImmediateVicinity.

Although the TAMRA that is used in the laboratory data of the bead-boundrelease monitor is a fluorophore, in other contexts, TAMRA can be aquencher. TAMRA acts as a quencher in TaqMan® probes that contain FAMand TAMRA.

Additional Accounts of Experimental Setup and Laboratory Data.

The Present disclosure provides data on controlled5(6)-Carboxytetramethylrhodamine (TAMRA) concentrations in phosphatebuffer (10 mM phosphate, 154 mM sodium, pH 8.0) within filledpico-wells, compartmentalized by air. Fluorescence images captured (10ms, 2 ms exposures) and well-area quantitated by mean pixel intensity(n≥100) to generate a concentration vs fluorescence intensitycalibration curve. The above data take the form of a standard curve,showing fluorescence at various predetermined concentrations of freeTAMRA (2, 10, 30, 60, 100 mM TAMRA). This standard curve was preparedunder two different conditions, that is, where the photographic imagewas taken with a 2 millisecond exposure or with a 10 millisecondexposure. The experiment used for preparing the standard curve wasconducted in picowells, but there were not any beads used in thisexperiment (just known amounts of TAMRA). The photographic image is notshown in this patent document, because the data merely take the form ofa standard curve, which may also be called a calibration curve.

The experimental setup included the following. For Scheme X),TentaGel-Lys(PCL1-Tamra)-QSY7 bead structure. QSY7 (gray) quenches theTamra fluorophore (orange) while covalently attached to bead through aphotocleavable linker (purple). Irradiation from UV (365 nm) providesquantitative release of compounds in situ

FIGS. 31A-31B discloses emission data resulting after catalytic actionof aspartyl protease on quencher-fluorophore substrate. Greaterfluorescence means that the enzyme is more catalytically active. Lesserfluorescence means that the enzyme is less catalytically active, thatis, there the enzyme is more inhibited by a free inhibitor, where theinhibitor was freed from a bead, and where freedom was obtained bycleavage of light-cleavable linker. Images were captured following UVrelease and Cathepsin-D assay incubation (λ_(ex)=480 nm, λ_(em)=525 nm).Wells containing positive control beads could be identified spectrallyby Cy5 fluorophore (λ_(ex)=645 nm, λ_(em)=665 nm, orange false color). Asection was analyzed with a line-plot across open well volume, Wellscontaining negative control beads elicit no Cathepsin-D inhibition.Assay volume within wells containing positive control beads are dark,indicating strong inhibition. Assay volume within empty wells iscomparable to wells containing negative control beads.

FIG. 32 illustrates the following procedure. Further regarding SchemeX), Picowell substrate (46 pL per well) is enclosed in a flowcell, wellswetted under vacuum, a suspension of TentaGel-Lys(PCL1-TAMRA)-QSY7 beadsare introduced, and air pulled across flow-cell, compartmentalizing eachwell (top). Flowcell is irradiated by a UV LED (λ_(mean) 365 nm) withcontrolled luminous flux, allowed to equilibrate (20 min), beforefluorescence microscopy images taken to quantitate released compound(TAMRA) concentration (bottom) (FIG. 32). In detail, FIG. 32 showsdrawings of cross-section of picowell, illustrating the steps wherepicowells wetted in a flowcell, the step where beads in a suspension areintroduced over the picowells, resulting in one bead per picowell, thestep of drawing air across flowcell in order to reduce excessivedispersion solution and resulting in a meniscus dropping below thesurface of the planar top surface of the picowell plate, the step ofcontrolled UV exposure (365 nm), resulting in release of some TAMRA, andthe step of provoking light emission from TAMRA with detectingfluorescent signal with fluorescent microscopy (excite 531/40 nm) (emit594/40 nm). The notation, “slash 40” refers to the bandwidth, that is,it means that cut-off filters confined the light to the range of: 531 nmplus 20 nm and minus 20 nm, and to 594 nm, plus 20 nm and minus 20 nm(this slash notation can be used for excitation wavelengths and also toemission wavelengths).

The present inventors acquired photographs showing the following data(see, FIGS. 33A-33F). Fluorescence emission (λ_(ex) 531/40 nm, λ_(em)593/40) of fluorophore (TAMRA) released from 10-μmTentaGel-Lys(PCL1-TAMRA)-QSY7 beads after UV LED (365 nm) exposure inpico-well flow cell. A) No significant emission above background priorto UV exposure (0 J/cm²), owed to the FRET quenching effect of QSY7.TAMRA release allowed to reach equilibrium (20 min) following UVexposures of (B) 25 J/cm², (C) 257 J/cm², (D) 489 J/cm², (E) 721 J/cm²,(F) 953 J/cm² then imaged using appropriate exposure times. Fluorescenceemission was measured within the volume surrounding each bead to measureTAMRA concentration (FIGS. 33A-33F) The notation, “slash 40” refers tothe bandwidth, that is, it means that cut-off filters confined the lightto the range of: 531 nm plus 20 nm and minus 20 nm (this slash notationcan be used for excitation wavelengths and also to emissionwavelengths).

The following is an interpretation, by the present inventors, of some ofthe fluorescence data from testing and use of the bead-bound releasemonitor (see, FIG. 34) Concentration of bead-released TAMRA insidepico-wells (45 pL) after UV exposure (365 nm). Image analysis used meanpixel intensity of the solution surrounding bead-filled wells (n≥14),normalized to image exposure time, then correlated to standard curve ofknown TAMRA concentrations in pico wells. Error bars represent 1σ,calculated from RSD %. UV released compound concentrations were 1.1 μM(RSD % 8.9), 54.3 μM (RSD % 5.2), 142 μM (RSD % 4.2), 174 μM (RSD %7.7), 197.3 μM (RSD % 10.1) (FIG. 34)

(XII) Biochemical Assays for Compounds (Assays that are not Cell-Based)

A variety of biochemical assays are possible using beads withinpicowells. Non-limiting examples include binding assays, enzymaticassays, catalytic assays, fluorescence based assays, luminescence basedassays, scattering based assays, and so on. Examples are elaboratedbelow.

Biochemical Assays that are Sensitive to Inhibitors of Proteases andPeptidases.

Where the goal is to detect and then develop a drug that inhibits aprotease, screening assay can use a mixture of a particular protease orpeptidase, a suitable cleavable substrate, and a color-based assay or afluorescence-based assay that is sensitive to the degree of inhibitionby candidate drug compounds. For example, one reagent can be abead-bound compound, where the compound has not yet been tested foractivity. Another reagent can take the form of bead-bound pepstatin (anestablished inhibitor of HIV-1 protease) (Hilton and Wolkowicz (2010)PLoS ONE. 5:e10940 (7 pages)). Yet another reagent can be a cleavablesubstrate of HIV-1 protease, and where cleavage by the HIV-1 proteaseresults in a change in color or a change in fluorescence.Positive-screening drug candidates are identified where a particularassay (in a given microwell) results in a difference in color (or adifference in fluorescence). The cleavable substrate takes the form of asusceptible peptide that is covalently bound to and flanked by aquencher and a fluorescers. Before cleavage, the fluorophore does notfluoresce, because of the nearby quencher, but after cleavage,fluorescence materializes (see, Lood et al (2017) PLoS ONE. 12:e0173919(11 pages); Ekici et al (2009) Biochemistry. 48:5753-5759; Carmona et al(2006) Nature Protocols. 1:1971-1976). The reagents and methods of thepresent disclosure encompass the above-disclosed technology.

Enzyme-Based Screening Assay for Compounds that Inhibit UbiquitinLigases, where the Reagents Include MDM2 (Enzyme) and p53 (Substrate).

Applicants have conducted working tests based on the followingtechnology. MDM2 regulates the amount of p53 in the cell. MDM2 isoverexpressed in some cancers. MDM2 is an enzyme, as shown by thestatement that, “In vitro studies have shown that purified MDM2 . . . issufficient to ubiquitinate . . . p53” (Leslie et al (2015) J. Biol.Chem. 290:12941-12950). Applicant's goal is to discover inhibitors ofMDM2, where these inhibitors are expected to reduce ubiquitination ofp53 and thus reduce subsequent degradation of p53. In view of theexpected increase in p53 in the cell, an inhibitor with the aboveproperty is expected to be useful for treating cancer.

Applicants used the following enzyme-based assay for assessing theinfluence of lenalidomide on ubiquitination of p53, as mediated byMDM2/HDM2. Applicants used reagents from the following kit: MDM2/HDM2Ubiquitin Ligase Kit—p53 Substrate (Boston Biochem, Cambridge, Mass.).One of the reagents used in the assay was a bead with a covalently boundantibody. The bead was TentaGel® M NH2 (cat. no. M30102, Rapp PolymereGmbH, Germany) and the antibody was anti-human p53 monoclonal antibody,biosynthesized in a mouse. MDM2 is an E3 ligase that can use p53 as asubstrate, where MDM2 catalyzes ubiquitination of the p53.

Goal of Activating p53 for Reducing Cancer.

A relation between MDM2, the transcription factor called, “p53,” andanti-cancer therapy is suggested by the following description. Thedescription is, “MDM2 is an E3 ubiquitin ligase that ubiquitinates p53,targeting it for proteasomal degradation” (Ortiz, Lozano (2018)Oncogene. 37:332-340). p53 has tumor-suppressing activity. p53 activitycan be inhibited by MDM2. According to Wu et al, MDM2 is a, “p53-bindingprotein” (see, Wu, Buckley, Chernov (2015) Cell Death Disease. 6:e2035). Where a compound prevents ubiquitination of p53, for example, byblocking interactions between MDM2 and p53, the compound might beexpected to function as an anti-cancer drug.

Goal of the Screening Assay.

A purpose of the screening assay is to discover compounds that influenceubiquitination of p53, for example, compounds that stimulate p53ubiquitination and compounds that inhibit p53 ubiquitination. In detail,the purpose is to discover compounds that are inhibiting or activating,where their effect is via MDM-2 and either E1 ligase, E2 ligase, or E3ligase. MDM2 means, “murine double minute.” MDM2 has been called an, “E3ubiquitin ligase.” When MDM2 occurs in the cell, evidence suggests itsactivity in catalyzing the ubiquitination of p53 requires a number ofother proteins, such as CUL4A, DDB1, and RoC1 (see, Banks, Gavrilova(2006) Cell Cycle. 5:1719-1729; Nag et al (2004) Cancer Res.64:8152-8155). Banks et al have described a physical interactioninvolving p53 and MDM2 as, “L2DTL, PCNA and DDB1/CUL4A complexes werefound to physically interact with p53 tumor suppressor and its regulatorMDM2/HDM2” (Banks, Gavrilova (2006) Cell Cycle. 5:1719-1729). Nag et alhave also described a physical interaction involving p53 and MDM2 as,“Cul4A functions as an E3 ligase and participates in the proteolysis ofseveral regulatory proteins through the ubiquitin-proteasome pathway.Here, we show that Cul4A associates with MDM2 and p53” (Nag et al (2004)Cancer Res. 64:8152-8155).

Desired Read-Out from the Bead-Based Assay for Modulators of p53Ubiquitination.

Where screening compounds results in a positive-screening hit, that is,where there is more AF488 fluorescence, this means that an ACTIVATOR hasbeen discovered. And where screening compounds results in apositive-screening hit, where there is a REDUCTION in fluorescence, thismeans that an INHIBITOR has been discovered. A compound that inhibitsubiquitination of p53, suggests that the compound can be used fortreating cancer. Also a compound that specifically inhibitsubiquitination of p53, that is, where the compound does not inhibitubiquitination of other proteins, or where the compound inhibitsubiquitination of other proteins with inhibition that is less severethan for p53, also suggests that the compound can be used for treatingcancer.

Materials.

Materials included E3 Ligase kit K-200B from Boston Biochem. BostonBiochem catalog describes this kit as: Mdm2/HDM2 Ubiquitin LigaseKit—p53 Substrate. The following concerns Mdm2, which is part of thiskit. This kit does not include cereblon. Lenalidomide and similarcompounds can bind to either cereblon or to Mdm2, where the end-resultis activation of ubiquitin ligase. Materials also included Diamond WhiteGlass microscope slides, 25 mm×75 mm (Globe Scientific, Paramus, N.J.).Corning Stirrer/Hot Plate (settings from zero to ten) 698 Watts, ModelPC-420. N-hydroxy-succinimide (NHS). Methyltetrazine (mTET).AlexaFluor488 (AF488) (ThermoFisher Scientific). TentaGel beads M NH2(cat. No. M30102) (Rapp Polymere GmbH). Parafilm (Sigma-Aldrich, St.Louis, Mo.). FIG. 8 shows the structure of Alexa Fluor® 488. Thestructure of Alexa Fluor 488 (AF488) is shown in Product Information forAlexaFluor488-Nanogold-Streptavidin (Nanoprobes, Inc., Yaphank, N.Y.).

(XIII) Cell-Based Assays for Chemical Compounds

Cell-based assays that are conducted in a picowell can use human cells,non-human cells, human cancer cells, non-human cancer cells, bacterialcells, cells of a parasite such as plasmodium cells. Also, cell-basedassays can be conducted with human cells or non-human cells that are“killed but metabolically active,” that is, where their genome has beencross-linked to allow metabolism but to prevent cell division (see, U.S.Pat. Publ. No. 2007/0207170 of Dubensky, which is incorporated herein byreference in its entirety). Moreover, cell-based assays can be conductedon apoptotic cells, necrotic cells, or on dead cells. Cell-based assayswith bacterial cells can be used to screen for antibiotics. Human cellsthat are infected with a virus can be used to screen for anti-viralagents. Combinations of cells are provided for cell-based assays. Forexample, combinations of dendritic cells and T cells are provided toscreen for and identify compounds that stimulate antigen presentationor, alternatively, that impair antigen presentation.

Cell-based assays can be based on a primary culture of cells, forexample, as obtained from a biopsy of normal tissue, a biopsy from asolid tumor, or from a hematological cancer, or from a circulating solidtumor cells. Also, cell-based assays can be based on cells that havebeen passaged one or more times.

Cell-based assays that are conducted in a picowell can use a culturethat contains only one cell, or that contains two cells, three cells,four cells, five cells, or about 2 cells, about 3 cells, about 4 cells,about 5 cells, or a plurality of cells, or less than 3 cells, less than4 cells, less than 5 cells, and so on.

Applicants have conducted working tests based on the followingtechnology. This describes cell-based assays for screening compound forthe exemplary embodiment where lenalidomide (test compound) inhibitsubiquitin-mediated proteolysis of a transcription factor. Thetranscription factors include Ikaros and Aiolos.

The present disclosure provides a cell-based assay that screenscompounds on a bead-bound compounds, and where screening is done with aplate bearing many picowells. The components of the cell-based assayinclude, a picowell for holding a bead-bound chemical library, whereeach bead has attached to it substantially only one, uniform type ofcompound. The compounds are released by way of a cleavable linker.Mammalian cells are cultured in the picowell. The picowell also includesculture medium. The presently disclosed non-limiting example withlenalidomide is a proof-of-principle example that can be used forscreening chemical libraries in order to discover other compounds thatmodulate ubiquitination of a given target protein.

Shorter Description of a Cell-Based Assay.

Recombinant cells are used as a reagent for detecting and screening forcompounds that induce proteolysis of green fluorescent protein (GFP),where the read-out that identifies a positively screening compound isthe situation where green-colored cells become colorless cells, or cellswith reduced green color. Regarding the mechanism of this cell-basedassay, the mechanism of action of lenalidomide in causing green-coloredcells become colorless cells, or cells with a reduced green color, isthat the lenalidomide binds to a protein called, “cereblon.” In thecell, cereblon is part of a complex of proteins called, “E3 ubiquitinligase.” Cereblon is the direct target of the anti-cancer drugs,lenalidomide, thalidomide, and pomalidomide. The normal and constitutiveactivity of E3 ubiquitin ligase, and its relation to cereblon, has beendescribed as, “cereblon . . . promotes proteosomal degradation [oftarget proteins] by engaging the . . . E3 ubiquitin ligase” (see, Akuffoet al (2018) J. Biol. Chem. 293:6187-6200). In contrast to the normalactivity of E3 ubiquitin ligase, when a drug such as lenalidomide,thalidomide, or pomalidomide is added, the result is that the,“lenalidomide, thalidomide, and pomalidomide . . . promote[s] theubiquitination and degradation of . . . substrates by an E3 ubiquitinligase . . . each of these drugs induces degradation of transcriptionfactors, IKZF1 and IKZF3” (Kronke et al (2015) Nature. 523:183-188).

Regarding terminology, cereblon has been described as being part of acomplex of proteins that is called, “E3 ligase” and also called, “E3ubiquitin ligase.” Generally, cereblon by itself is not called an “E3ligase. The following excerpts reveal how the word “cereblon” is used.According to Akuffo et al (2018) J. Biol. Chem. 293:6187-6200, “Uponbinding to thalidomide . . . the E3 ligase substrate receptor cereblon .. . promotes proteosomal destruction [of the substrate] by engaging theDDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase.” Consistently, Yang et al(2018) J. Biol. Chem. 293:10141-10157, discloses that, “Cereblon . . .functions as a substrate receptor of the cullin-4 RING E3 ligase tomediate protein [the substrate] ubiquitination.” Zhu et al (2014) Blood.124:536-545, state that, “Thalidomide binds CRBN [cereblon] to alter thefunction of the E3 ubiquitin ligase complex . . . composed of CRBN,DDB1, and CUL4.” Lopez-Girona et al (2012) Leukemia. 26:2326-2335, statethat, “studies identified E3 ligase protein cereblon (CRBN) as a directmolecular target . . . of thalidomide . . . CRBN and . . . DDB1 form afunctional E3 ligase complex with Cul4A and Roc1.”

To view the big picture of the cell-based assay devised and used by theApplicants, the first step is that lenalidomide is added to cells. Thelast step is that IKZF1 and IKZF3 are degraded. Where IKZF1 occurs as afusion protein with GFP, then the last step is that the entire fusionprotein is degraded by the proteasome. Similarly, where IKZF3 occurs asa fusion protein with GFP, then the final step is that this entirefusion protein gets degraded by the proteasome. The result of GFPdegradation is that the cell, which was once green-fluorescing cell, isturned into a non-fluorescing cell.

Longer Description of a Cell-Based Assay.

This concerns names of proteins of E3 ubiquitin ligase (a complex ofproteins), names of proteins that bind to this complex, and names ofproteins that are the target of this complex. For these names, thepublished literature is not consistent. Sometimes it refers to theprotein by the name of the protein, and sometimes it refers to theprotein using the name of the gene that encodes the protein. For thisreason, the following account uses the protein name together with thegene name, such as “cereblom” (name of protein” and “CRBN” (name ofgene). Also, “Ikaros” is the name of a protein, while the gene's name isIKZF1. Also, “Aiolos” is the name of a protein, IKZF3 is the name of thegene. “Cullin-ring finger ligase-4” is the name of a protein, and thegene's name is CRL4. “Regulator of cullin-1” is the name of a protein,and the gene's name is ROC1. ROC1 is also known as, RBX1 (Jia and Sun(2009) Cell Division. 4:16. DOI:10.1186. “Cullin-4A” is the name of aprotein and the gene's name is CUL4A. See, Schafer, Ye, Chopra (2018)Ann. Rheum. Dis. DOI:10.1136; Chen, Peng, Hu (2015) Scientific Reports.5:10667; Matyskiela et al (2016) Nature. 535:252-257; Akuffo et al(2018) J. Biol. Chem. 293:6187-6200).

E3 ubiquitin ligase catalyzes the transfer of a residue of ubiquitin toa target protein, where the consequence is that the target protein getssent to the proteasome for degradation. The E3 ligase catalyzesattachment of ubiquitin to one or more lysine residues of the targetprotein. Humans express about 617 different E3 ubiquitin ligase enzymes(see, Shearer et al (2015) Molecular Cancer Res. 13:1523-1532). E3ubiquitin ligase is a complex of these proteins: DNA damage bindingprotein-1 (DDB1); Cullin-4 (CUL4A or CUL4B); Regulator of Cullins-1(RoC1); and RING Box-domain protein (RBX1). As stated above, RoC1 is thesame protein as RBX1 (see, Jia and Sun (2009) Cell Division. 4:16. DOI:10.1186). When cereblon (CRBN) joins the E3 ubiquitin ligase complex,the resulting larger complex is called: CRL4^(CRBN)(Matyskiela et al(2016) Nature. 535:252-257). The term “CRL4” means, “Cullin-4 RINGLigase” (Gandhi et al (2013) Brit. J. Haematol. 164:233-244; Chamberlainet al (2014) Nature Struct. Mol. Biol. 21:803-809). The abovediscrepancies in nomenclature need to be taken into account when readingthe literature of cereblon.

The following are longer versions of the short excerpts disclosed above.Shown below is yet another form of nomenclature, namely, the term:“CRL4^(CRBN) E3 ubiquitin ligase.” The longer account more fullyintegrates the various names and cellular events. “The relation betweencereblon (CRBN) and E3 ubiquitin ligase complex has been described as,“cereblon (CRBN) promotes proteosomal degradation [of target protein] byengaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase” (Akuffo et al(2018) J. Biol. Chem. 293:6187-6200). Regarding anti-cancer drugs,“lenalidomide, thalidomide, and pomalidomide . . . promote theubiquitination and degradation of . . . substrates by an E3 ubiquitinligase. These compounds bind CRBN, the substrate adaptor for theCRL4^(CRBN) E3 ubiquitin ligase . . . each of these drugs inducesdegradation of . . . transcription factors, IKZF1 and IKZF3” (Kronke etal (2015) Nature. 523:183-188).

This concerns cell-based assays where any given microwell, nanowell, orpicowell contains a bead where bead has covalently linked compounds,where the compound is attached via a cleavable linker, and where thewell contains one or more cultured mammalian cells. Responses tocompounds and to drug candidates of the present disclosure can beassessed by way of one or more biomarkers.

Biomarkers include diagnostic biomarkers, biomarkers that predict if agiven patient will respond (get better) to a given drug, and biomarkersthat predict if a given patient will experience unacceptable toxicity toa given drug (Brody, T. (2016) Clinical Trials: Study Design, Endpointsand Biomarkers, Drug Safety, and FDA and ICH Guidelines, 2^(nd) ed.,Elsevier, San Diego, Calif.). The present disclosure makes use of yetanother kind of biomarker, namely, a biomarker that monitors response ofa patient to a given drug, after drug therapy has been initiated. Togive an example, the following concerns the biomarker “peroxiredoxin6(PRDX6) and lung cancer. According to Hughes et al, “PRDX6 levels incell media from . . . cell lines increased . . . after gefitinibtreatment vs. vehicle . . . PRDX6 accumulation over time correlatedpositively with gefitinib sensitivity. Serum PRDX6 levels . . .increased markedly during the first 24 hours of treatment . . . changesin serum PRDX6 during the course of gefitinib treatment . . . offers . .. advantages over imaging-based strategies for monitoring response toanti-EGFR agents.” Please note comment that the biomarker has advantagesover a more direct measure of efficacy of response, namely, use of“imaging” to detect decrease in tumor size and numbers (Hughes et al(2018) Cancer Biomarkers. 22:333-344). Other biomarkers that monitorresponse to anti-cancer drugs include CA125 for monitoring response toplatin therapy for ovarian cancer, and serum HSPB1 for monitoringresponse to chemotherapy with ovarian cancer (see, Rohr et al (2016)Anticancer Res. 36:1015-1022; Stope et al (2016) Anticancer Res.36:3321-3327).

Cytokine Expression.

Responses can be assessed by measuring expressed cytokines, such asIL-2, IL-4, IL-6, IL-10, IFN-gamma, and TNF-alpha. These particularcytokines can be simultaneously measured using gold nanostructuresbearing antibodies that specifically recognize one of these cytokines,where detection involves plasmon resonance (Spackova, Wrobel, Homola(2016) Proceedings of the IEEE. 104:2380-2408; Oh et al (2014) ACS Nano.8:2667-2676). Cytokines expressed by single cells, such as a single Tcell, can be measured by way of fluorescent antibodies, in a device thatincludes microwells (Zhu, Stybayeva (2009) Anal. Chem. 81:8150-8156).The above methods are useful as reagents and methods for the presentdisclosure.

In some embodiments, antibodies to cytokines may be attached to thewalls of the picowells, wherein any cytokines released, ordifferentially released, from cells, as a function of drug exposure canbe captured by the antibodies bound to the walls of the picowells. Thecaptured cytokines may be identified by a second set of labeledantibodies. In some embodiments, antibodies for cytokines may beattached to capping beads. The capping beads may then be embedded in acrosslinking hydrogel sheet that may be peeled off and subjected tofurther analysis, for example, via ELISA, mass spectrometer or otheranalytical techniques.

Apoptosis.

Real-time data on apoptosis, and early events in apoptosis of singlecells can be measured with Surface-Enhanced Raman Spectroscopy (SERS)and with Localized Surface Plasmon Resonance (LSPR) (see, Stojanovic,Schasfoort (2016) Sensing Bio-Sensing Res.7:48-54; Loo, Lau, Kong (2017)Micromachines. 8:338. DOI:10.3390). Stajanovic, supra, detects releasefrom cells of cytochrome C, EpCam, and CD49e. Loo et al, supra, measuresrelease from cell of cytochrome C, where detection involves a DNAaptamer (this DNA aptamer works like an antibody). Zhou et al detectearly apoptosis in single cells using SERS, where what is measured isphosphatidyl serine on the cell membrane (see, Zhou, Wang, Yuan (2016)Analyst. 141:4293-4298). In addition to collecting data on apoptosis,SERS can be used for assessing drug activity by collecting data onstages of mitosis, release of metabolites, expression of a biomoleculebound to the plasma membrane (see, Cialla-May et al (2017) Chem. Soc.Rev. 46:3945-3961). Plasmon resonance can measure protein denaturationand DNA fragmentation that occurs in apoptosis (see, Kang, Austin,El-Sayed (2014) ACS Nano. 8:4883-4892). Plasmon resonance (SERS) candistinguish between cancer cells and normal cells, by measuring thepercentage of mitotic proteins in the alpha helix form versus in betasheet form (Panikkanvalappil, Hira, El-Sayed (2014) J. Am. Chem. Soc.136:159-15968). The above methods are suitable as reagents and methodsfor the present disclosure.

Apoptosis can also be measured in cultured cells in a method not usingplasmonic resonance, but that instead uses immunocytochemistry usinganti-cleaved caspase-3 antibody (Shih et al (2017) Mol. Cancer Ther.16:1212-1223).

General Information on Cell-Based Assays.

Cell-based assays of the present disclosure can be used to testresponses from human cancer cells, cells from a solid tumor, cells froma hematological cancer, human stem cells, human hepatocytes, apathogenic bacterium, an infectious bacterium, human cells infected witha bacterium, human cells infected with a virus, and so on. The assayscan detect morphological response of the cell, such as migration, aswell as genetic responses and biochemical responses.

Assays of the present disclosure can be designed to detect response ofcells that are situated inside a microwell, or to detect response ofcells that are situated outside a microwell, such as in a nutrientmedium situated as a layer above the array of microwells. Also, assaysof the present disclosure can be designed to detect responses of cells,where cells and beads are situated within a medium, where cells aresituated within a medium and beads are above or below the medium, wherecells are situated on top of a medium and where beads are situated aboveor within or below the medium.

The present disclosure provides a population of cells to a microwellarray. In embodiments, at least about 5%, at least about 10%, at leastabout 20%, at least about 40%, at least about 60%, at least about 80%,at least about 90%, at least about 95%, or at least about 100%, of thepopulation of cells resides inside the microwells (and not in any regionsituated above the microwells). In embodiments, the proportion of cellsthat resides inside of the wells, with the rest being situated in alayer of nutrient medium residing above the array of wells, can be about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, about 100%, or in any range defined bytwo of these numbers, such as the range of “about 60% to about 90%.”

Matrix for Cells.

For assays of biological activity of cells, and where cells are exposedto compounds released from beads, or where cells are exposed tobead-bound compounds, suitable matrices include those that include oneor more of the following: poly-D-lysine (PDL), poly-L-lysine (PLL),poly-L-ornithine (PLO), vitronectin, osteopontin, collagen, peptidesthat contain RGD sequence, polypeptides that contain RGD sequence,laminin, laminin/fibronectin complex, laminin/entactin complex, and soon. Suitable matrices also include products available from Corning,Inc., such as, PuraMatrix® Peptide Hydrogel®, Cell-Tak® cell and tissueadhesive, Matrigel®, and so on. See, Corning Life Sciences (2015)Corning Cell Culture Surfaces, Tewksbury, Mass. (20 pages), De Castro,Orive, Pedraz (2005) J. Microencapsul. 22:303-315. In exclusionaryembodiments, the present disclosure can exclude any composition ormethod that includes one of the above matrices or one of the abovepolymers.

In embodiments, the present disclosure provides an array, whereindividual microwells contain a bead, one or more cells, and either asolution (without any matrix) or a matrix or a combined solution andmatrix. The matrix can be a hydrogel, polylysine, vitronectin,MatriGel®, and so on.

Activity of bead-bound compounds or of bead-released compounds can beconducted. Assays to assess activity can include, activating orinhibiting an enzyme, activating or inhibiting a cell-signaling cascadeor an individual cell-signaling protein, binding to an antibody (or to acomplementary determining region (CDR) of an antibody, to a variableregion of an antibody), inhibiting the binding of a ligand or substrateto an enzyme (or to an antibody, or to a variable region of anantibody).

For the above assays, the readout can be determined with fluorescenceassays, for example, involving a fluorophore linked to a quencher (F-Q).The linker can be designed to be cleavable by an endoprotease, DNAse,RNAse, or phosopholipase (see, Stefflova, Zheng (2007) FrontiersBioscience. 12:4709-4721). The term “molecular beacon” refers to thistype of F-Q molecule, however, “molecular probe” has also been used torefer to constructs where separation of F and Q is induced byhybridization, as in TaqMan® assays (Tyagi and Kramer (1996) NatureBiotechnol. 14:303-308; Tsourkas, Behlke, Bao (2003) Nucleic Acids Res.15:1319-1330).

Transcriptional Profiling in Response to Drug Exposure.

The DNA barcodes of this disclosure may be modified to containresponse-capture elements, where the response capture elements capturethe response of cells to perturbations encoded by the encoding portionsof the barcode. In some embodiments, the DNA barcodes may terminate in apoly-T section (multiple repeats of the thymidne nucloetide), where thepoly-T sequence may be used to capture poly-A terminated mRNA moleculesreleased from lysed cells. In some embodiments, the response-capturesequence may be complementary to genes of interest, thereby capturingthe expression profile of desired genes via hybridization to the beadsof this embodiment. In some embodiments, picowells may contain a singlecell picowell whose transcriptional profile is captured on the bead. Insome other embodiments, a plurality of cells may be contained in thepicowell whose transcriptional profile is being captured.

In one exemplary workflow, the following procedure may be followed tocapture transcriptional response of cells to drugs. (a) Picowellsdesigned to capture single cells per well are provided. (b) Acompound-laden, DNA barcoded bead is introduced into the picowells, suchthat one bead is present per picowell. (c) Compounds are released fromthe beads in each picowell by appropriate methods (UV treatment forcompounds attached via UV cleavable linker, diffusion in case of beadssoaked in compounds, acid cleavable, base cleavable, temperaturecleavable etc., as appropriate for the beads of the embodiment). (d) Thepicowells may be isolated from each other via a capping bead thatretains contents within the picowell or by other means such as an airbarrier or an oil barrier on top of the picowells. (e) The cells in thepicowells are allowed to incubate in the presence of the compoundsreleased from the beads for a duration. (f) After a suitable amount oftime, say 1 hr, 2 hrs, 5 hrs, 9 hrs, 12 hrs, 15 hrs, 18 hrs, one day, 3days, one week, two weeks, one months, or another appropriate time basedon the assay, the cells are lysed by a lysing method. The lysing methodsmay involve addition of detergents, repeated cycles of freezing andthawing, heating, addition of membrane disrupting peptides, mechanicalagitation or other suitable means. (g) once lysed, the contents of thecell are exposed to the bead within the picowell, at which time theresponse-capture elements on the beads of the picowell are enabled tocapture the response they are designed for. In some embodiments, theresponse capture are poly-T sequences which capture the complete mRNAprofile of the cell (or cells) within each picowell. In someembodiments, the response-capture elements are designed to capturespecific DNA or RNA sequences from the cell. In some embodiments, thetranscriptional response of the cell may be captured as a function ofdosage (or concentration) of compounds.

EXAMPLES Example 1. First Workflow

The present disclosure provides methods, including that outlined belowas “First Workflow” and as “Second Workflow.”

The First Workflow includes the steps: (1) Generate DELB, (2) Beads intopicowells, (3) Load assay reagents into picowells, (4) Releasebead-bound compounds, (5) Measure assay readout, (6) Rank the assayreadout, and (7) Generate a new set of DELBs.

Generate DELB.

First, create the DNA encoded library on beads (DELB). Each beadcontains a population of the exact, same compound, though slightdepartures from this may occur where some of the manufactured compoundshad incomplete couplings or were suffered chemical damage, such asinadvertent oxidation.

Beads into Picowells.

Then, deposit beads in picowells. In a preferred embodiment, eachpicowell gets only one bead. Each picowell can have a round upper edge,a round lower edge, a solid circular bottom, an open top, and a wall.The wall's bottom is defined by the round upper edge and by the roundlower edge. In a preferred embodiment, the wall is angled, where thediameter of the round upper edge is greater than the diameter of theround lower edge. In this way, the wall (viewed by itself) resembles aslice of an inverted cone. The picowell array can be prepared, so thatthere is a redundancy of beads. In other words, the array can beprepared so that two of the beads, out of the many thousands of beadsthat are placed into the picowells, contain exactly the same compound.The redundancy can be, e.g., 2 beads, 3 beads, 4 beads, 5 beads, 10beads, 20 beads, 40 beads, 60 beads, 80 beads, 100 beads, and so on, orabout 2, about 3, about 4, about 10, about 20, about 40, about 60, about80, about 100, about 200, about 500, about 1,000 beads, and so on, ormore than 2, more than 5, more than 10, more than 20, more than 40, morethan 60, more than 80, more than 100, more than 200, more than 500, morethan 1,000 beads, and so on.

Load Assay Reagents into Picowells.

Introduce reagents into each picowell that can be used to assessbiochemical activity of each bead-bound compound. The biochemicalactivity can take the form of a binding activity, enzyme inhibitionactivity, enzyme activation activity, activity of a living mammaliancell (where the molecular target is not known), activity of a livingmammalian cell (where the molecular target is known), and so on. Thereagent can take the form of a FRET reagent plus an enzyme. The FRETreagent can be a fluorophore linked by way of a protease substrate to aquencher. The enzyme can be a substrate of that protease, which iscleavable by the protease. The bead-bound compound is being tested forability to inhibit the protease.

After loading assay materials, each picowell can be capped by a film, ormany or all of the picowells can be capped by one film, or many or allof the picowells can be capped by a film with pimples where each pimplefits into a picowell, or or where each picowell is fitted with a poroussphere. In embodiments, about 5% of the volume about 10% of the volume,about 20% of the volume, about 30% of the volume, or about 40% of thevolume of the sphere fits into the picowell (where the remainder isflush with the surface or resides above the surface). In embodiments,about 5%, about 10%, about 20%, about 40%, about 60%, about 80%, about90%, or about 100% of the pimple fits into the picowell.

Release Bead-Bound Compounds.

Perform a step that causes release of the bead-bound compound. Inembodiments, the step can cause release of about 0.1%, about 0.2%, about0.1%, about 0.2%, about 2%, about 5%, about 10%, about 20%, about 40%,about 60%, about 80%, about 99%, or about 100% of the compounds that areattached to a given bead. Release can be effected by light, by achemical reagent, by an enzyme, by a shift in temperature, by anycombination thereof, and so on.

Release can Take the Form of:

(i) Single release, (ii) Multiple release, (iii) Continual release.Multiple release, for example, can take the form of several emissions ofultraviolet light, where each emission is sufficient to cleave about 10%of the bead-bound compound that happens to be attached to the bead atthe start of that light emission. Continual release, for example, cantake the form of continual emission of light over the course of onehour, resulting in a steadily increasing concentrations of freecompound. In this situation, the steadily increasing concentrations offree compound (cleaved compound) may be for the purpose of titrating thetarget of that compound. A titration experiment of this kind can be usedto assess potency of a given compound. To provide non-limiting examples,with a single release method, a period of light exposure is followed bya subsequent period where readout is taken, and with a continual releasemethod, light exposure continues during some, most, or all of the periodwhere readout is taken.

In exclusionary embodiments, the present disclosure can exclude anymethod, reagent, composition, or system that uses single release, thatuses multiple release, or that uses continual release.

Measure Assay Readout.

Detect the above-disclosed biochemical activity, and the influence ofthe released compound on that activity. This biochemical activity cantake the form of enzymatic activity, activity of a reporter gene,genetic activity (e.g., rate of transcription or translation), bindingactivity (e.g., antigen to antibody), cellular activity (e.g., change inmigration, change in cell-signaling pathway, change in morphology).Activity can be detected by fluorescence, chromogenic activity,luminescence, light microscopy, TaqMan® assays, molecular beacons, massspectrometry, Raman spectroscopy, Localized Surface Plasmon Resonance(LSPR), Surface Plasmon-Coupled Emission (SPCE), Surface-Enhanced RamanScattering (SERS), and so on. Detection can be with methods that aretotally remote, such as fluorescence detection or light microscopy or,alternatively, by methods that involve taking a sample from thepicowell. In one embodiment, a sample that contains a mixture ofreactants and products can be withdrawn for analysis by way of aspherical porous sponge that is partially inserted into one of thepicowells.

Rank the Assay Readout.

In this step, assay readouts from a plurality of different compounds(each type of compound associated with one particular bead), are rankedin terms of their ability to activate, inhibit, or in some way tomodulate the biochemical activity.

Generate a New Set of DELBs.

The steps that are described above inform the user of various compoundsthat exhibit a biochemical activity. The information may take the formof one compound with maximal activity, with the rest having about halfmaximal activity or less. Alternatively, the information may take theform of several compounds having a similar maximal activity, with theother compounds having about half maximal activity or less. A new set ofDELBs can be created as follows. One or more of the highest-rankingcompounds (the lead compounds) can be used as a basis for manufacturinga new set of DELBs, based on one or more of the following non-limitingstrategies: (i) Replacing an aliphatic chain with a homolog, such asreplacing a propanol side chain with a butanol side chain; (ii)Replacing an aliphatic chain with an isomer, such as replacing apropanol side chain with an isopropanol side chain; (iii) Replacing apeptide bond with an analog of a peptide bond, such as with a bond thatcannot be hydrolyzed by peptidases; (iv) Replacing one type of chargedgroup with another type of charged group, such as replacing a phosphategroup with a phosphonate, sulfate, sulfonate, or carboxyl group.

Example Two. Second Workflow

The Second Workflow involves picowells that are sealed with caps. Thecaps can take the form of spheres of slightly greater diameter than thediameter of the picowells, where this diameter is measured at the toprim of the picowell (not measured at the bottom of the picowell). Thecap can be made to fit snuggly into the top of the picowell bysubjecting the entire picowell plate to mild-gravity centrifugation. InSecond Workflow, the caps take the form of beads that contain linkers,where each linker is linked to a compound. The linkers are cleavablelinkers, where cleavage released the compounds and allows them todiffuse to the cells. This type of cap is called an “active cap.” TheSecond Workflow includes the steps, (1) Generate DELB, (2) Load assayreagents into picowells, (3) Cap picowells with DELB, (4) Releasebead-bound compounds from the bead that acts as a cap, (5) Measure assayreadout, (6) Determine sequence of the DNA barcode that is on the bead;(7) Rank the assay readout, and (8) Generate a new set of DELBs.

Example 3. Release Control

This concerns controlling and monitoring release of bead-boundcompounds. Applicants devised the following procedure for synthesizingbead-bound release-monitor. See, FIG. 11 and the following text.

FIG. 11 describes steps in the organic synthesis of the above exemplaryembodiment of a bead-bound release-monitor.

Step 1. Provide the Resin

TentaGel® resin (M30102, 10 μm NH2, 0.23 mmol/g, 10 mg; MB 160230, 160μm RAM, 0.46 mmol/g, 2 mg) was weighed into a tube (1.5 mL Eppendorf)and swelled (400 μL, DMA).

Resin was transferred into fritted spin-column (MoBiCol® spin column,Fisher Scientific), solvent removed through filter by vacuum, andpendent Fmoc was deprotected (5% Piperazine with 2% DBU in DMA, 400 μL;2×10 min at 40° C.). The MoBiCol spin column has a 10 micrometer largefrit and a luer-lock cap.

Resin was filtered over vacuum, and washed (2×DMA, 400 μL; 3×DCM, 400μL; 1× DMA, 400 μL).

Step 2. Couple Lysine Linker to Resin

A solution was prepared containing L-Fmoc-Lys(Mtt)-OH (21 moles, 6.6eq.), DIEA (42 moles, 13.3 eq.), COMU (21 μmoles, 6.6 eq.) mixed in DMA(350 μL), incubated (1 min, RT), then added to dry resin inside thefritted spin-column, vortexed, and incubated (15 min, 40° C.) to amidatethe free amine. Resin was filtered by vacuum, and this reaction wasrepeated, once.

Resin was filtered over vacuum, and washed (2×DMA, 400 μL; 3×DCM, 400μL; 1× DMA, 400 μL).

Step 3. Remove the Fmoc Protecting Group

The pendent Fmoc was deprotected (5% Piperazine with 2% DBU in DMA, 400μL; 2×10 min at 40° C.).

Resin was filtered over vacuum, and washed (2×DMA, 400 μL; 3×DCM, 400μL; 1× DMA, 400 μL).

Step 4. Couple the Quencher

A solution was prepared containing QSY7-NHS (4.9 moles, 1.55 eq.), Oxyma(9.5 eq, 3.3 eq.), DIC (21 μmoles, 6.6 eq.), TMP (3.5 μmoles, 1.1 eq.)mixed in DMA (350 μL), incubated (1 min, RT), then added to dry resininside the fritted spin-column, vortexed, and incubated (14 hr, 40° C.)to amidate the free amine.

Resin was filtered over vacuum, and washed (2×DMA, 400 μL; 3×DCM, 400μL; 1× DMA, 400 μL).

A solution was prepared containing Acetic Anhydride (80 moles, 25.3eq.), TMP (80 moles, 25.3 eq.), mixed in DMA (400 μL), mixed then addedto dry resin inside the fritted spin-column, vortexed, and incubated (20min, RT)

Resin was filtered over vacuum, washed (2×DMA, 400 μL; 3×DCM), andincubated in DCM (1 hr, RT), then filtered over vacuum and dried invacuum chamber (30 min, 2.5 PSI)

Step 5. Remove the Mtt Protecting Group

Mtt deprotection cocktail was prepared containing TFA (96 μL), Methanol(16 μL), mixed in DCM (1488 μL) giving 6:1:93% of TFA:Methanol:DCMsolution.

Mtt deprotection cocktail was added to the fully dried resin (400 μL),mixed, eluted by filtration over vacuum, then sequential aliquots of Mttdeprotection cocktail (4×400 μL) were added, mixed, incubated (5 min,RT), and eluted for a combined total incubation time of 20 min at RT.

Resin was filtered over vacuum, and washed (3×DCM, 400 μL; 1×DMA, 400μL; lx DMA with 2% DIEA, 400 μL; 3×DMA, 400 μL).

Step 6. Couple the Photocleavable Linker to Epsilon-Amino of Lysine

A solution was prepared containing Fmoc-PCL-OH (32 moles, 10 eq.), Oxyma(32 moles, 10 eq.), DIC (50 moles, 15.8 eq.), TMP (32 moles, 10 eq.)mixed in DMA (400 μL), incubated (1 min, RT), then added to dry resininside the fritted spin-column, vortexed, and incubated (14 hr, 40° C.)to amidate the free ε-amine.

Resin was filtered over vacuum, and washed (2×DMA, 400 μL; 3×DCM, 400μL; 1× DMA, 400 μL).

Step 7. Remove the Fmoc Protecting Group from the Previously CoupledPhotocleavable Linker

The pendent Fmoc was deprotected (5% Piperazine with 2% DBU in DMA, 400μL; 2×10 min at 40° C.).

Resin was filtered over vacuum, and washed (2×DMA, 400 μL; 3×DCM, 400μL; lx DMA, 400 μL).

Step 8. Couple the Fluorophore

A solution was prepared containing TAMRA (6 moles, 1.9 eq.), TMP (24moles, 7.6 eq.), COMU (16 moles, 5 eq.), mixed in DMA (400 μL),incubated (1 min, RT), then added to dry resin inside the frittedspin-column, vortexed, and incubated with mixing (2 hr, 40° C., 800 RPM)to amidate the free amine.

Resin was filtered over vacuum, and washed (2×DMA, 400 μL; 3×DCM, 400μL; 2× DMA, 400 μL; 2×DMSO), then incubated with mixing in DMSO (16 hr,40° C.).

The following provides a broader account of the above-disclosedlaboratory procedures.

Bi-Functional Linker Attached to Bead.

Bi-functional linker was synthesized in solution and attached to anamine-functionalized beads. FIG. 11 discloses pathway of organicsynthesis, starting with lysine. Lysine-Boc was than connected by TCOlinker. The main part of the linker was took the form of polyethyleneglycol (PEG) with a nitrogen at one end. Boc was a leaving group in thisconnecting reaction. The TCA that was used was actually a racemate ofhydroxy-TCO. The hydroxyl group of this TCO derivative was connected toa carbon atom located four carbon atoms away from one side of the doublebond (this is the same thing as being located three carbon atoms awayfrom the other side of the double bond). As shown in FIG. 11, the firstproduct in the multi-step synthesis took the form ofBoc-lysine-linker-TCO. The hydroxyl group that was once part ofhydroxy-TCO is still attached to the TCO group, where it is situated inbetween the aminated-polyethylene glycol group and the TCO group (FIG.11).

The second set in the synthetic pathway involved treatment with HCl andaddition of a photocleavable linker (PCL). The product of this secondstep was the same as the product of the first step, except with the Bocgroup replaced with the photocleavable linker. The lysine moiety takes acentral position in the product of the second step. Regarding the lysinemoiety, this lysine moiety has a free carboxyl group, and in the thirdstep of the procedure, an aminated bead is connected to this freehydroxyl group, resulting in the synthesis of a bead-bound reagent,where the reagent takes the form of two branches, and where at the endof one branch is a TCO tag, and where at the end of the other branch isan aromatic ring bearing a cleavable bond. To attached a chemicalmonomer to the distal end of the photocleavable linker, first the Fmocgroup is removed, and here the Fmoc group is replaced with a hydrogenatom.

Removing Fmoc.

According to Isidro-Llobet et al, “Fmoc . . . is removed by bases mainlysecondary amines, because they are better at capturing thedibenzofulvene generated during the removal” (Isidro-Llobet et al (2009)Chem. Rev. 109:2455-2504). Alternatively, Fmoc can be removed bycatalytic hydrogenolysis with Pd/BaSO₄, or by liquid ammonia andmorpholine or piperidine.

Removal of Fmoc Group Followed by Attaching a Chemical Monomer.

Applicants then condensed a chemical monomer having a carboxylic acidgroup, where the result was generation of an amide bond. (This step notshown in any figure.)

Example 4. Cereblon-Based Assay for Active Compounds

Results from Cell-Based Assays of Compounds (Cereblon-Based Assay).Reagents and Methods for Cell-Based Assay.

Applicants used CCL-2 HeLa cells obtained from ATCC (American TypeCulture Collection, Manasses, Va.). Cell medium was Gibco DMEM highglucose medium buffered with HEPES. Atmosphere above cell culture wasatmospheric air supplemented with 5% carbon dioxide, with the incubatorat 37 degrees C. Cell medium was DMEM plus 10% fetal bovine serum,supplemented with GlutaMAX® (Gibco Thermofisher), and also supplementedwith non-essential amino acids and penicillin plus streptomycin (GibcoThermofisher, Waltham, Mass.). HeLa cells were transfected with aconstruct taking the form of LTR-CTCF-Promoter-IKZF1 (orIKZF3)-mNeon-P2A-mScar-LTR-CTCF. mScarlet is an element used as apositive control. mScarlet encodes red fluorescent protein called,“mScarlet” (see, Bindels et al (2017) Nature Methods. 14:53-56). Thepromoter is doxycycline indudicble promoter, which enables rapid onsetinduction and titration of the substrate. P2A is an element situated inbetween two other polypeptides. P2A functions, during translation, toproduct two separate polypeptides, thus allowing the mScar polypeptideto function as a positive control that produces red light, without beinginfluenced by ubiquitination and degradation of the fusion proteinconsisting of IKZF1/Green Fluorescent Protein (GFP). mNeonGreen isderived from the lancelet Branchiostoma lanceolatum multimeric yellowfluorescence protein (Allele Biotechnology, San Diego, Calif.). P2A is aregion that allows self-cleaving at a point in the P2A protein. Moreaccurately, the P2A peptide causes ribosomes to skip the synthesis ofthe glycyl-prolyl peptide bond at the C-terminus of a 2A peptide,leading to the cleavage between a 2A peptide and its immediatedownstream peptide (Kim, Lee, Li, Choi (2011) PLoS ONE. 6:e18556 (8pages).

Demonstration of Efficacy of Cell-Based Assay for Test Compounds.

The following demonstrates use of a cell-based assay for test compoundstaking the form of lenalidomide and analogus of lenalidomide. FIGS.5A-5H disclose results from HeLa cells that were transfected withlentiviral vector, where the vector expressed Green Fluorescent Protein(GFP) and a red fluorescent protein (mScarlet). Increasing theconcentration of added lenalidomide resulted in progressively less greenfluorescence, and elimination of green fluorescence at highestconcentrations. But lenalidomide did not substantially decrease redfluorescence. Top: Expression of IKZF1/GFP fusion protein. Bottom:Expression of mScarlett control. Lenalidomide was added at zero, 0.1,1.0, or 10 micromolar.

FIGS. 6A-6H disclose results from HeLa cells that were transfected withlentiviral vector, where the vector expressed Green Fluorescent Protein(GFP) and red fluorescent protein (mScarlet). Increasing concentrationof added lenalidomide resulted in progressively less green fluorescence,and elimination of green fluorescence at highest concentrations. Butlenalidomide did not substantially decrease red fluorescence. Top:Expression of IKZF3/GFP fusion protein. Bottom: Expression of mScarlettcontrol. Lenalidomide was added at zero, 0.1, 1.0, or 10 micromolar.

To summarize the pathway where lenalidomide causes proteolysis of thefusion proteins, first lenalidomide is added to the HeLa cells. Then,the lenalidomide binds to the cereblon that naturally occurs in thesecells. This cereblon occurs in a complex with E3 ubiquitin ligase. E3ubiquitin ligase responds to the lenalidomide by tagging the recombinantIKZF1 fusion protein (or the recombinant IKZF3 fusion protein) withubiquitin. The end-result is that the ubiquitin-tagged fusion protein isdegraded in the cell's proteasome.

Coating the Picowell Plates.

This describes solutions that are applied to the top surface of apicowell plate, but that do not necessarily enter and coat inside ofpicowells. This is also about solutions that are applied to the topsurface of a picowell plate and that enter the picowells, and that coatthe bottom surface of the picowells. Applicants added a solution ofPluronic® 127 (Sigma Aldrich, St. Louis, Mo.) to dry plastic. The resultis a surface that is hydrophilic, and no longer hydrophobic. Then, thesurface was washed with water. Then, phosphate buffered saline (PBS) wasadded, where this PBS enters inside the picowells. Moving air is appliedby way of a vacuum, where the result is that it causes small bubbles inthe picowells to expand, and where the bubbles are then replaced withthe PBS, and where the end result is that much of the picowell getsfilled with PBS. Then, PBS was replaced with vitronectin coatingsolution (AF-VMB-220) (PeproTech, Rocky Hill, N.J.). Pluronics® 127 is:H(OCH₂CH₂)_(x) (OCH₂CHCH₃)_(y) (OCH₂CH₂)_(z)OH. After applying thevitronectin coating solution, Applicants incubated for 30 min at 37degrees C. to allow the coating solution to get into picowells. ThePluronic 127 coats the ridges that separate the picowells, and thevitronectin is at bottom of picowells. HeLa cells attach to vitronectinand when they attach to the vitronectin, they adhere to the bottom ofthe picowell.

HeLa cells were screened for successfully transfected cells by way offlow cytometry. Two criteria were used simultaneously for determiningsuccessful transfection. First, lenalidomide was added to cell media 2days before sorting by flow cytometry. A positive cell was that whichwas red-plus and green-minus, where red-PLUS meant that the cells weretransfected with the gene encoding mScar, and where green-MINUS meantthat the lenalidomide had in fact promoted the ubiquitination anddegradation of the fusion protein, IKZF1/mNeon (or the fusion protein,IKZF3/mNeon). Regarding doxycycline, doxycycline was used at 3micromolar in order to induce expression of the lentiviral vectorconstruct. A concentration/induction curve with doxycycline is shown byGo and Ho (2002) J. Gene Medicine. 4:258-270). After transfection withthe lentivirus vector, the following condition was used to keep IKZF1minimally expressed in growing cells. The condition was to leavedoxycycline out of the medium, and also to use “insulating sequences” inthe construct. The insulating sequences prevent read-through frompromoters outside of the construct. Insulating sequences have beendescribed (see, Anton et al (2005) Cancer Gene Therapy. 12:640-646; Carret al (2017) PLoS ONE. 12:e0176013). Insulating sequences preventpromoters that are outside of the construct from driving expression ofan open reading frame (ORF) that is part of the construct. To put cellsinto picowells, cells can be transferred to the top surface of apicowell plate, at a given ratio of, [number of cells]/[number ofpicowells]. The ratio can be, for example, about 1 cell/40 wells, about1 cell/20 wells, about 1 cell/10 wells, about 2 cells/10 wells, about 4cells/10 wells, about 8 cells/10 wells, about 16 cells/10 wells, about32 cells/10 wells, about 50 cells/10 wells, about 100 cells/10 wells,and so on. The cells can be used for assays in picowells as soon ascells attach to the vitronectin that coats the bottom of the picowell.

Details of Lentivirus Construct and Cell Culture.

This concerns constructing reporter cell lines for IKZF1/3, culturingthem in picowells, and assaying them with bulk lenalidomide. Theplasmids carrying reporter construct were assembled from parts usingGibson assembly (see maps attached). Lentivirus with reporter construct,as well as UbC driven rtTA-M2.2 were made in LentiX HEK293T cells(Clontech, Palo Alto, Calif.) with 3^(rd) generation packaging system(chimeric CMV promoter and no tat protein). The plasmids weretransfected via calcium precipitation method. Virus supernatant washarvested in the recommended LentiX media plus 1% bovine serum albumin(BSA), and filtered through 0.45 um low protein bind filters(Millipore). The host HeLa cells were obtained from ATCC, cultured instandard conditions. Viral supernatant was applied to sub-confluent HeLaculture, after 24 hours changed to LentiX media with Doxicyclin. Twodays before clone selection, lenalidomide was added to the culture.Clones were selected via fluorescence activated cell sorting (FACS),gated on both AlexaFluor 488 (negative) and Cy3 channels (positive).Clones were grown for 10 days without lenalidomide before assays. Themost stable expression level clones are used for screening.

This describes experiment to seal cells with beads and lyse cellsthrough porous beads. 96 well plate with picowell patterned bottom(MuWells) is treated with Pluronic F127 detergent (Sigma-Aldrich, St.Louis, Mo.) without vacuum applied to passivate upper part of the wells.After 30 min incubation, excess of detergent is washed away withphosphate buffered saline (PBS) or distilled H₂O. Wells are flushed withethanol and dried in the biosafety cabinet with the air flow. Wells arewetted with PBS under strong vacuum to a completion, and PBS is replacedwith Virtonectin coating reagent (Preprotech). The plate is incubatedfor 30 min at 37 C. Vitronectin coating reagent is removed and reportercells are seeded at desirable density. From the moment of cell seeding,media stays in the dish throughout the assay. TentaGel® beads carryingthe photocleavable compound could be seeded before vitronectin coating,or after cell seeding. PEG polymer beads are loaded on top of theculture in the excess over the well number. Spin the plate at 400 rcffor 1 min. Photo-release the compound off the beads using 365 nm LEDlight source for appropriate amount of time. Incubate in the CO₂incubator until the imaging (readout of the fluorescent reporters).

Constructs.

FIG. 20 and FIG. 21 disclose the relevant constructs. Each of thesefigures discloses the sequence that is to be integrated into the HeLacell genome, and each of the figures discloses the carrier sequence (thesequence belonging to lentivirus). Sequence belonging to lentivirus isfrom about one o'clock to about nine o'clock, where this sequenced isbracketed by two long terminal repeats (LTRs). Sequence from about nineo'clock to about one o'clock gets integrated into HeLa cell genome. Indetail, first a plasmid is transfected into producer cells (HEK93T)(Clontech, Palo Alto, Calif.). The producer cells produce and thenrelease lentivirus. The released lentivirus then infects HeLa cells andintegrates nucleic acids into the HeLa cell genome.

Optics.

For the present cell culture experiments, Applicants used EBQ100Isolated mercury lamp connected to HBO 100 (Carl Zeiss Microscopy, GmbH,Germany), which was connected to an Axiovert 200-M Carl Zeiss microscopewith Ludl Electronic Products stage (Ludl Electronic Products, Ltd.,Hawthorne, N.Y.). Applicants also used filter cubes with mercury lamp,where filter cubes controlled wavelength of excitation and alsocontrolled wavelength of detecting emission. Images were captured withBasler ACA2440-35UM (Basler AG, 22926, Ahrensburg, Germany). Halogenlamp was used, as an alternative to mercury lamp. Microwell plates,picowell plates, and the like, were held in place with a plate holderand an “XY stage” with controller. XY stages and other precisepositioning stages for optics use are available from, Newmark Systems,Inc., Rancho Santa Margarita, Calif.; Aerotech, Inc., Pittsburgh, Pa.,Physik Instrumente GmBH, 76228 Karlsruhe, Germany.

Example 5. MDM2-Based Assay for Active Compounds

Modifying Glass to Contain an Amino Group.

Silica substrates can be modified to contain an amino group, by way ofone or more of a number of “functional silanes.” These “functionalsilanes” are 3-aminopropyl-triethoxysilane (APTES),3-aminopropyl-trimethoxysilane (APTMS),N-(2-aminoethyl)-3-aminopropyltriethoxysilane (AEAPTES),N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPTMS), andN-(6-aminohexyl)aminomethyltriethoxysilane (AHAMTES). Reactions of thesereagents with glass can be conducted in a vapor phase or in a solutionphase (see, Zhu, Lerum, Chen (2012) Langmuir. 28:416-423).

Results from Biochemical Assays of Compounds (MDM2-Based Assay).Laboratory Methods.

The following reagent was applied to a glass slide. The glass slide wasmodified to have amino groups. The reagent was NHS-PEG-mTET. NHS isN-hydroxy-succinimide. NHS is a type of activated ester. NHS is usefulin bioconjugation reactions, such as surface activation of microbeads orof microarray slides (Klykov and Weller (2015) Analytical Methods.7:6443-6448).

PEG is polyethylene glycol. mTET is methyltetrazine. This reagent wasmixed with DMSO, and then a volume of 2 microliters was applied to theglass slide. The mixture was made by mixing 10 microliters of 50 mMNHS-PEG-mTET with 30 microliters DMSO. The NHS group reacts with theamino groups of the glass side, where the result is that the mTET groupis affixed to the glass slide. The goal of the mTET was to create acovalent link between the slide and the bead.

TCO and tetrazine can mediate “click chemistry” reactions. Examples ofthese click chemistry reactions, is using antibodies that arefunctionalized with tetrazine to couple with DNA that is functionalizedby TCO. Or using antibodies modified with TCO to couple withtetrazine-modified beads (see, van Buggenum et al (2016) ScientificReports. 6:22675 (DOI:10.1038); Rahim et al (2015) Bioconjug. Chem.18:352-360; Haun et al (2010) Nature Nanotechnol. 5:660-665).

In detail, the glass slide was prepared by applying a sheet of parafilmto the top of the slide, where the parafilm had an aperture cut out ofthe middle, where the drop of the above mixture was applied in theaperture directly to the glass slide. Before applying the mixture, theglass slide with the parafilm on top was heated at full heat for 90seconds, in order to create a tight seal between the parafilm and theslide, in order to prevent seepage of liquids after applying the mixtureto the open area (the aperture) in the parafilm. The glass slide, withthe 2 microliter droplet sitting in the aperture cut into the Parafilm,was incubated overnight at room temperature. During the incubation, theglass slide was inside a petri dish, where the dish was covered with aglass cover that covered the top and sides of the petri dish. Before theovernight incubation, a square of Parafilm was placed over the drop andover the surrounding Parafilm, in order to prevent water fromevaporating from the drop.

Inventive Method to Make Complex of Slide/Bead/Antibody.

Applicants' method used beads that were functionalized by TCO. The TCOgroups of the bead mediated covalent attachment of themethyltetrazine-functionalized slide to the bead. Also, the TCO groupsof the bead mediated covalent attachment of themethyltetrazine-functionalized anti-p53 antibody to the bead.

Applicants surprisingly found that, if the first step is to contactslide and bead, then subsequent addition of antibody will NOT result incovalent attachment of the antibody to the bead. Also, Applicantssurprisingly found that, if the first step is to contact bead withantibody, then subsequent transfer of this mixture to the slide will NOTresult in covalent attachment of the bead to the slide. In a preferredmethod, all of these three reagents—the slide, the bead, and theantibody—are simultaneously brought into contact with each other. Inanother preferred embodiment, the bead and antibody are first mixedtogether to initiate covalent linking of the bead to the antibody, andthen immediately or within a few minutes, this mixture is applied to theslide, where the result is covalent linking of the bead to the slide.

Nature of the Enzyme-Based Screening Assay.

The assay takes the form of a glass slide with an attached bead. Thebead contains attached antibodies that are specific for binding to thetranscription factor, p53. This antibody can bind to human p53 and alsoto ubiquitinated human p53. So far, it can be seen that the assay methodinvolves a sandwich between the following reagents:

Slide/Covalently Bound Bead/Bead-Bound Anti-p53 Ab/Ubiquitinated p53

The readout from this assay is ubiquitinated-p53, where theubiquitinated-p53 is detected by a fluorescent antibody that is specificfor ubiquitin. In detail, the antibody is a polyclonal antibody made inthe goat, where the antibody is tagged with a fluorophore (AF488). FIG.8 discloses the structure of AF488. This fluorescent antibody binds toubiquitin. Thus, when ubiquitinated-p53 is detected, what exists is thefollowing sandwich:

Slide/Covalently Bound Bead/Bead-Bound Bound Anti-p53 Ab/Ubiquitinatedp53/Fluorescent Ab

Example 6. Sequencing DNA in Picowells

Sequencing of bead-bound DNA barcodes was performed, where beads weresituated in a picowell, one bead per picowell. The assay method involvedinterrogating each position on the bead-bound DNA barcode, one at atime, by way of transient binding of fluorescent nucleotides. Each beadcontained about one hundred attomoles of coupled DNA barcode, wherecoupling was by click-chemistry. This number is equivalent to aboutsixty million oligonucleotides, coupled per bead. For each base on theDNA barcode, the assay involves adding all four fluorescent dNTPs at thesame time. Without implying any limitation, the four fluorescent dNTPswere AF488-dGTP, CY3-dATP, TexasRed-dUTP, and CY5-dCTP. Fluorescentsignals were captured, and then processed by ImageJ software (NationalInstitutes of Health, NIH), to provide a corresponding numerical value.The data are from sequencing five consecutive nucleotides (all in a row)that was part of the bead-bound DNA barcode. The bead-bound DNA barcodeincluded a DNA hairpin region. The bases in the DNA hairpin regionannealed to itself, resulting in the formation of the hairpin, and wherethe 3′-terminal nucleotides in this DNA hairpin served as a sequencingprimer. Sequencing by transient binding was initiated at this3′-terminus. The sequencing assay was performed in triplicate, that is,using three different beads, where one DNA barcode sequence was used foreach of the three beads. In other words, each of the three beads wasexpected to provide a sequencing read-out identical to that provided bythe other two beads.

FIG. 28 discloses sequencing results, where sequencing was conducted onbead-bound DNA barcode. What is shown are results from interrogating thefirst base, the second base, the third base, the fourth base, and thefifth base. For each of these bases, what is separately shown, by way ofseparate histogram bars, is the fluorescent emission produced withinterrogation with AF488-dGTP, CY3-dATP, TexasRed-dUTP, and CY5-dCTP,respectively. Each of the four histogram bars has different graphics:AF488-dGTP (black outline, gray interior), CY3-dATP (black outline,white interior), TexasRed-dUTP (solid black histogram bar), and CY5-dCTP(solid gray histogram bar). The bead diameter was 10-14 micrometers,after swelling in aqueous solution. The volume of the picowell was 12picoliters.

The template sequence that was interrogated was:5′-CTCACATCCCATTTTCGCTTTAGT-3′ (SEQ ID NO: 1). For this particularsequencing assay, five consecutive bases were interrogated, where thefluorescent dNTPs that gave the biggest fluorescent signal werefluorescent dGTP, dATP, dGTP, dUTP, and dGTP, which corresponds to asequence on the template that is dC, dT, dC, dA, and dC. Thus, thesequencing results were 100% accurate. The results demonstrate that thebead-bound DNA barcodes can be sequenced, that is, when the DNA barcodeis still bound to the bead. In other words, the bead-bound DNA barcodesare sequencable.

Example 7. Cell Barcoding

Introduction to the Concept of Barcoding.

This introduces the concept of barcoding. A common barcoding techniqueis barcoding the transcriptome of a given single cell. FIG. 36 and FIG.37 illustrate steps for procedures where the transcriptome is capturedand amplified, in preparation for future sequencing. FIG. 36 shows lysisof cells to release mRNAs, followed by reverse transcription. FIG. 37shows capture of mRNAs by way of immobilized poly(dT), followed byreverse transcription, and finally sequencing. Sequencing can be withNext Generation Sequencing (NGS).

Some or most of the messenger RNA (mRNA) molecules from a given cell canbe tagged with a common barcode, where this tagging allows theresearchers to determine, for any given mRNA sequence, the origin ofthat coding sequence in terms of a given cell. For example, wherenucleic acids representing each of the separate transcriptomes from onehundred different single cells are mixed together, and where the nucleicacids from each of the 100 different single cell has its own barcode,then the following advantage will result. The advantage is that nucleicacids from all of the transcriptomes can be mixed together in one testtube, and then subjected to Next Generation Sequencing, where thebarcode enables the user to identify which information is from the samecell.

The above advantage is described in a different way, as follows. Inusing mRNA barcoding, a given single cell is processed so thatinformation from some or most of the mRNA molecules from that cell areconverted to corresponding molecules of cDNA, where each of these cDNAmolecules possesses exactly the same DNA barcode. This barcodingprocedure can be repeated with ten, twenty, 100, several hundred, orover 1,000 different cells, where the cDNA molecules from each of thesecells is distinguished by having a unique, cell-specific barcode. Thismethod enables the researcher to conduct DNA sequencing, all in onesequencing run, from a pool of all of the barcoded cDNA molecules fromall of the cells (all barcoded cDNA molecules mixed together, prior tosequencing) (see, Avital, Hashimshony, Yanai (2014) Genome Biology.15:110).

Barcodes that Tag Nucleic Acids Compared with Barcodes that Tag thePlasma Membrane.

Guidance is available for preparing libraries of chemicals, where eachchemical, or where all members of each class of chemicals, is associatedwith a unique DNA barcode (see, Brenner and Lerner (1992) Proc. Nat'l.Acad. Sci. 89:5381-5383; Bose, Wan, Carr (2015) Genome Biology. 16:120.DOI 10.1186). With the above barcoding example in mind, the followingprovides another type of barcoding which can also be applied to aparticular, single cell. The present disclosure provides cell-associatedbarcoding that takes the form of a tag that is stably attached to thecell's plasma membrane.

Option of at Least Two Kinds of Barcodes that Get Attached to the PlasmaMembrane-Bound.

A barcode used for tagging the plasma membrane of given cell can includea first barcode that identifies the type of cell, and a second barcodethat identifies a perturbant that was exposed to the cell. For example,the first barcode can identify the cell as originating from a healthyhuman subject, Human Subject No. 38 from Clinical Study No. 7, a humanprimary colorectal cancer cell line, a five-times passaged human primarycolorectal cancer cell line, a multiple myeloma human subject withmultiple myeloma, a treatment-naive Human Subject No. 23 with multiplemyeloma, or from a treatment-experienced Human Subject No. 32 withmultiple myeloma.

Also, the barcode can identify a “perturbant” that was given to thatparticular single cell (given either before or after barcoding). The“perturbant” can be an anti-cancer drug, a combination of anti-cancerdrugs, a combinatorially generated compound, or a combination of anantibody drug and a small molecule drug. The barcoding can be used tokeep track of a given single cell, and can be used to correlate thatcell with subsequent behaviors such as activation or inhibition with oneor more cell-signaling pathways, increased or decreased migration,apoptosis, necrosis, change in expression of one or more CD proteins(CD; cluster of differentiation), change in expression of one or moreoncogenes, change in expression of one or more microRNAs (miRNAs).Expression can be in terms of, transcription rate, level of a givenpolypeptide in the cell, change in location of a given protein fromcytosolic to membrane-bound, and so on.

Tagging Cell-Surface Oligosaccharides of Membrane-Bound Glycoproteins.

Methods and reagents are available for connecting tags, such as DNAbarcodes, to the plasma membrane of a living cell. Tagging can beaccomplished with a reagent consisting of a covalent complex of a DNAbarcode with a reactive moiety that attacks and covalently binds tooligosaccharide chains of membrane-bound glycoproteins. The literatureestablishes that hydrazide biocytin can be used to connect biotin tocarbohydrates on membrane-bound glycoproteins. The present disclosureuses this reagent, except with the biotin replaced with a DNA barcode.The carbohydrate needs to be oxidized to form aldehydes. The hydrazidereacts with the aldehyde to form a hydrazine link. The sialic acidcomponent on the oligosaccharides is easily oxidized with 1 mM Nameta-periodate (NaIO4). In conducting the oxidation step, andhydrazide-linking step, buffers with a primary amine group should beavoided. See, for example, “Instructions. EZ-LinkHydrazide Biocytin.Number 28020. ThermoScientific (2016) (4 pages), Bayer (1988) Analyt.Biochem. 170:271-281; Reisfeld (1987) Biochem. Biophys. Res. Commun.142:519-526, Wollscheid, Bibel, Watts (2009) Nature Biotechnol.27:378-386.

Another method for tagging the oligosaccharide moiety of glycoproteinson living cells, is to use periodate oxidation and aniline-catalyzedoxime ligation. This method uses mild periodate oxidation of sialicacids and then ligation with an aminoxy tag in the presence of aniline.In a variation of this method, galactose oxidase can be used tointroduce aldehydes into terminal galactose residues and terminalN-acetylgalactosamine (GalNAc) residues of oligosaccharides. Galactoseoxidase catalyzes the oxidation, at carbon-6, to generate an aldehyde.Following aldehyde generation, one can couple with aminoxybiotin usinganiline-catalyzed ligation (see, Ramya, Cravatt, Paulson (2013)Glycobiology. 23:211-221). The present disclosure replaces the biotinwith a DNA barcode and provides aniline-catalyzed ligation of anaminoxy-DNA barcode.

Tagging Mediated by an Antibody Bound to the Cell Surface.

The present disclosure provides methods and reagents for attachingbarcodes to the plasma membrane of a cell, where attachment is mediatedby an antibody that specifically binds to a membrane-bound protein. Theantibody can be covalently modified with trans-cyclooctene (TCO) wherethis modification can be conducted with an overnight incubation at 4degrees C. (see, Supporting Information (5 pages) for Devaraj, Haun,Weissleder (2009) Angew. Chem. Intl. 48:7013-7016). This covalentmodification of antibody can be carried out with the reagent,trans-cyclooctene succinimidyl carbonate (Devaraj, Haun, Weissleder(2009) Angew. Chem. Intl. 48:7013-7016). The antibody-tetrazine complexcan then be contacted with a cell, resulting in membrane-boundantibodies. The membrane-bound antibodies each bear a tetrazine moiety,which enables tagging of the antibody via click chemistry, such as, byexposing the antibodies to a DNA barcode-tetrazine complex.

Tetrazine can be introduced at free amino groups of the antibody, usingthe reagent, N-hydroxysuccinimide ester (NHS) (see, van Buggenum,Gerlach, Mulder (2016) Scientific Reports. 6:22675). Once the antibodycontains one or more tetrazine groups, the antibody can be furthermodified by attaching a DNA barcode, by way of a reagent that is TCO-DNAbarcode. With this modified antibody in hand, the antibody can then beused for a tagging living cell, where the antibody binds to amembrane-bound protein of the cell.

A complex of tetrazine-DNA barcode can be prepared. This complex canthen be introduced into a cell medium, where the medium includes cells,and where the cells bear the attached antibody-TCO complex. Where thetetrazine-DNA barcode contacts the membrane-bound antibody-TCO complex,the result is a click chemistry reaction where the cells become taggedwith the DNA barcode. This click chemistry reaction can be carried outfor 30 minutes at 37 degrees C.

Preferred antibodies for use in the above procedure are those that bindtightly and specifically to membrane-bound proteins of the plasmamembrane, where the membrane-bound protein occurs in high abundance, forexample, at over 50,000 copies per cell membrane, and where themembrane-bound protein is stable on the cell surface and does not muchrecycle into the cell's interior, and where the membrane-bound membranedoes not much shed into the culture medium.

Tagging Membrane-Bound Proteins with Azide Followed by Click Chemistrywith an Octyne Conjugate.

Azide can be introduced on membrane-bound proteins of a living cell byway of the enzyme, lipoic acid ligase, followed by attachment of afluorinated octyne compound that is conjugated to a DNA barcode. Theconjugation of a fluorinated octyne compound to a fluorophore isdescribed (see, Jewett and Bertozzi (2010) Chem. Soc. Rev. 39:1272-1279;Fernandez-Suarez, Bertozzi, Ting (2007) Nature Biotechnol.25:1483-1487). To reiterate, “Ting and co-workers introduced azides intomammalian cell-surface proteins using . . . lipoic acid ligase . . .[t]he protein could then be labeled with a fluorinatedcyclooctyne-conjugated fluorescent dye-conjugated fluorescent dye”(Jewett et al, supra).

Example 7. Caps Over Picowells

Capping Picowells.

Each picowell was capped with a sphere, one sphere to each picowell,where the sphere fits into the aperture (top opening) of the picowell.To apply the spheres to the picowell plate, the spheres are put intogrowth media and suspended, then applied to the top surface of thepicowell plate, and the sphere allowed to settle. Then, the entire plateis placed in a centrifuge and spun at a low-gravity, in order to get afirm sitting of the spheres in the aperture of each picowell.

Active Caps and Passive Caps.

FIG. 18A shows an active cap inserted into the top of a picowell, andFIG. 18B shows a passive cap inserted into the top of a picowell.Preferably, the caps are made of material that is softer than thematerial used to make the picowell plate, where the result is slightdeformation of the cap when it is pressed into the aperture of thepicowell, and where the result is a snug fit that prevents leakage. Inembodiments, the present disclosure provides one or more of active caps,passive caps, or both active caps and passive caps. Each cap may befree-standing and not connected to any other cap. In an alternativeembodiment, to more caps may be connected together, for example, by wayof a sheet of polymer that is capable of being layed upon the topsurface of the plate, and where a plurality of caps protrude from thebottom of the sheet of polymer, and where the protruding caps arepredeterminedly spaced in order to fit into each picowell. An active capmay be used instead of a bead that is capable of sitting on the floor ofa microwell. The active cap contains many attached copies ofsubstantially identical compounds, where each compound is attached tothe active cap (shown here in the sample of a spherical bead), and wherecleavage results in release of the compounds into the solution thatresides in the microwell (FIG. 18A).

Regarding the Passive Cap, the Passive Cap is Porous and it Acts Like aSponge.

It absorbs products from biochemical reactions, and thus facilitatescollection of products where the goal of the user is to determine theinfluence of a given compound on living, biological cells that arecultures in the picowell. In other words, the compound stimulates thecells to respond, where the response takes the form of increased (ordecreased) expression of one or more metabolites, and where some of themetabolites diffuse towards the passive cap and are absorbed by thepassive cap. The user can then collect the passive caps and analyze themetabolites that had absorbed to the passive cap (FIG. 18B).

Polymer Mat that Adheres to an Array of Caps.

FIGS. 19A-19D illustrate a polymer mat that is capable of adhering toeach cap in an array of porous caps. Once adhered, the polymer mat canbe peeled away and removed, bringing with it each porous cap in thearray. As a result, the polymer mat with the porous caps can be used forassays that measure metabolites or other chemicals that are associatedwith the porous cap.

To provide a step-wise example, each well in an array of many thousandsof picowells can contain one bead, where each bead contains one type ofcompound, where the compound is attached via a cleavable linker. Thepicowell also contains a solution as well as cultured cells. Thepicowell is sealed with a porous cap, and where the porous cap contactsthe solution and is able to capture (sample; absorb; absorb) metabolitesthat are released from the cultured cells. The metabolites can bemetabolites of the compound, or the metabolites can take the form ofcytokines, interleukins, products of intermediary metabolism, microRNAmolecules, exosomes, and so on. Finally, a solution of polyacrylamide ispoured over the picowell plate, and the polyacrylamide allowed to soakinto the thousands of porous caps, and then solidify in the form of amat that is firmly adhered to each and every one of the caps. Thesolidified mat is then removed, where each cap is separately analyzedfor absorbed metabolites.

In preferred embodiments, a polyacrylamide gel is used to crosslink thecapping beads into the enmeshing layer or the mat. The protocol tocreate an 20% solution of polyacrylamide solution that can be pouredover the picowell array to cure and enmesh the capping bead is asfollows. Add 4 ml of a 40% bis-acrylamide solution and 2 ml of 1.5 MTris pH 8.8 to 1.8 ml distilled deionized water. Just before pouringthis mixture over the capped picowell array, 80 microliters of the freeradical initializer ammonium persulfate (APS, 10% stock solution), and 8microliter of the free radical stabilizerN,N,N′,N′-tetramethylethylene-diamine (TEMED) are added to begincrosslinking of the gel. The gel layer is poured before completecrosslinking and allowed to fully crosslink over the capped picowellarray. One fully crosslinked (stiff enough to be handled, or roughly 60minutes of setting), the polyacrylamide layer may be peeled off usingtweezers. It is found that the capping beads are lifted off the tops ofthe picowells and get attached to the polyacrylamide layer. Thisbehavior can be observed for multiple bead types includingpolyacrylamide beads, Tentagel beads, polystyrene beads and silicabeads.

Measuring Efficacy of Cap in Preventing Leaks.

In embodiments, the efficacy of a cap can be determined by using thebead with the photocleavable linker. Images of a picowell, or of severalpicowells in one particular picowell array can be captured just beforeexposing picowells to UV light, and in the time frame after exposingpicowells to UV light. For example, images can be captured at t=minusten seconds and at t=10 seconds, 20 sec, 40 sec, 60 sec, 2 minutes, 4min, 8 min, 15 min, 60 min, 90 min, 2 hours, 3 hours, and 4 hours.Excellent efficacy can be shown where the fluorescence of a given wellat 2 hours is equal to at least 90%, at least 95%, at least 98%, orabout 100% the fluorescence found at t=10 seconds, with subtraction ofthe background image taken at t=minus ten seconds. Images can also betaken of a region of the picowell plate outside of the picowell, forexample, in the immediate vicinity of the cap. Excellent efficacy can beshown where the fluorescence of an area on the surface of the plate(outside of the picowell) and in the immediate vicinity of the cap isless than 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than0.01%, less than 0.005%, or less than 0.001%. This comparison may bemade without regard to the volume of the fluid in the well, and withoutregard to the volume of any fluid situated on top of the plate andoutside of the cap, and here, the comparison may simply take intoaccount the entire visual field that is captured by the light detector.Alternatively, the comparison may be made with correction of the depthof the fluid (depth of picowell; depth of fluid on top of the picowellplate). Also alternatively, the comparison may take into accountdiffusion of any leaking fluorophore over the entire surface of thepicowell plate.

How Barcoding Fits into the Reagents and Methods of the PresentDisclosure.

The following provides further embodiments of the reagents and methodsof the present disclosure.

Reagents and Capabilities.

A microscopic bead is provided. The microscopic bead can be covalentlymodified by a plurality of first linkers, each capable of coupling byway of solid-phase synthesis with monomers, where completion of thesolid-phase synthesis creates a member of a chemical library. Thismember of the chemical library is bead-bound. The same microscopic beadcan be covalently modified by a plurality of second linkers, eachcapable of being coupled with a plurality of DNA barcodes. This memberof the DNA barcode is bead-bound.

Example 9. DNA Barcode of the Present Disclosure

This concerns a set of information that can be printed on paper, orstored in computer language, that provides a “DNA barcode” thatcorrelates a DNA sequence with a chemical library member. This DNAbarcode may be called a “legend” or a “key.” The DNA barcode alsoprovides nucleic acids that can identify a specific class of chemicalcompounds, such as analogs of a specific FDA-approved anti-cancer drugs,or that can identify the user's name, or that can identify a specificdisease that is to be tested with the bead-bound chemical library.

Example 10. Lenalidomide Analogs

FIGS. 13, 14, and 15 disclose the conversion of lenalidomide to threedifferent derivatives, each derivative bearing a carboxylic acid group.Each of these carboxylic acid groups can subsequently be used tocondensed with the bead-linker complex. In this situation, where thecarboxylic acid group is condensed to the bead-linker complex, it isattached at the position that was previously occupied by Fmoc.

Starting with a Primary Amine and Converting it to a Carboxylic Acid(FIG. 13).

Applicants take the approach of generating a library of compounds byconverting a compound with a primary amine to a compound with a carboxylgroup. FIG. 13 discloses starting with lenalidomide. Lenalidomide has aprimary amine. To this is added, succinic anhydride in4-dimethylaminopyridine (DMA) and acetonitrile (ACN). The succinicanhydride condenses with the primary amino group, resulting inlenalidomide bearing a carboxylic acid group. The term “cat.” in thefigure means, catalytic.

Subsequently, this carboxylic acid group can be linked to a bead. Thus,the resulting complex is: BEAD-succinic acid moiety-lenalidomide

FIG. 14 discloses starting with linalidomide and addingt-butyl-bromoacetate, to give an intermediate. The intermediate is thentreated with FmocOSu (o-succinimide), to produce a final product that isa carboxylic acid derivative of lenalidomide. The carboxylic acid moietycan then be condensed with a free amino group, for example, with thefree amino group that once had an attached Fmoc group. Alternatively,the carboxylic acid can be condensed with the free amino group of achemical monomer residing on the bead, where the result of thecondensation is two chemical monomers attached to each other.

FIG. 15 discloses lenalidomide as the starting material. Thelenalidomide is reacted with 3-carboxybenzaldehyde, where the aldehydegroup condenses with the amino group, resulting in yet another type ofcarboxylic acid derivative of lenalidoimide.

FIG. 16A, FIG. 16B, and FIG. 16C discloses yet another approach ofApplicants for generating a library of novel and unique bead-boundcompounds, where compounds can be released from the bead, and thentested for activity in cell-based assays or in cell-free assays. Each ofthe three compounds is a lenalidomide analogue, where the primary amineis in a unique position of the benzene ring.

Example 11. Picowells Containing Cells Together with Beads that have aCoupled Response-Capture Element

The present disclosure provides reagents, systems, and methods forassessing response of a cell to a compound, and where response that ismeasured takes the form of changes in the transcriptome. “Changes in thetranscriptome” can refer, without implying any limitation, to change inamount each and every type of unique mRNA in the cell, and well as tochange in amount of a pre-determined set of mRNA molecules in the cell.“Changes in transcriptome” includes change from below the lower limit ofdetection to becoming detectable, as well as change from beingdetectable to dropping below the lower limit of detection, where thesechanges are associated with release of the bead-bound compound.

Cells can be lysed by adding detergent or surfactant to the picowellarray. For example, a volume of buffer containing detergent can bepipetted into a microwell that contains, within it, many thousands ofpicowells. The detergent can be allowed to diffuse into all of thepicowells, causing lysis of the cells within, release of mRNA, andfinally binding by the bead-bound “capture response element.”

Cell Lysis.

Cells can be lysed by one or more cycles of freezing and thawing (Bose,Wan, Carr (2015) Genome Biology. 16:120. DOI 10.1186). Cells can also belysed with perfluoro-1-octanol with shaking (Macosko, Basu, Satija(2015) Cell. 161:1202-1214; Ziegenhain (2017) Molecular Cell.65:631-643; Eastburn, Sciambi, Abate (2014) Nucleic Acids Res. 42:e128).Also, cells can be lysed by a combination of a surfactant (Tween-20®)and a protease (Eastburn, Sciambi, Abate (2013) Anal. Chem.85:8016-8021). Lysis of cells results in release of mRNA. The mRNA iscaptured by the bead that resides in the same picowell as the lysed cell(or cells). The bead contains a huge number of bead-boundpolynucleotides, where each polynucleotide contains two nucleic acid,where the first nucleic acid contains a common DNA barcode and thesecond nucleic acid contains a “response capture element.” Where thegoal is indiscriminate capture of all mRNAs in the cell, the “responsecapture element” can take the form of poly(dT). This poly(dT) binds tothe poly(A) tail of the mRNA molecules.

More Cell Lysis Conditions.

Cell lysis can be effected by exposure to detergent with a sodium salt,for example, 0.05% Triton X-100 with 15 mM NaCl, 25 mM NaCl, 50 mM NaCl,75 mM NaCl, 100 mM NaCl, 0.1% Triton X-100 with 15 mM NaCl, 25 mM NaCl,50 mM NaCl, 75 mM NaCl, 100 mM NaCl, 0.2% Triton X-100 with 15 mM NaCl,25 mM NaCl, 50 mM NaCl, 75 mM NaCl, 100 mM NaCl, or 0.5% Triton X-100with 15 mM NaCl, 25 mM NaCl, 50 mM NaCl, 75 mM NaCl, 100 mM NaCl, orwith detergent with a potassium salt, such as, 0.05% Triton X-100 with15 mM KCl, 25 mM KCl, 50 mM KCl, 75 mM KCl, 100 mM KCl, 0.1% TritonX-100 with 15 mM KCl, 25 mM KCl, 50 mM KCl, 75 mM KCl, 100 mM KCl, 0.2%Triton X-100 with 15 mM KCl, 25 mM KCl, 50 mM KCl, 75 mM KCl, 100 mMKCl, or 0.5% Triton X-100 with 15 mM KCl, 25 mM KCl, 50 mM KCl, 75 mMKCl, 100 mM KCl. Exposure can be for 10 min, 20 min, 40 min, or 60 minat about 4 degrees C., or at room temperature (23 degrees C.), and soon.

Present Disclosure can Assess Influence of a Compound on an ExpressionProfiles.

A bead-bound capture element can take the form of one or moredeoxyribonucleotides that can specifically hybridize to one or more mRNAmolecules of interest, where the one or more mRNA molecules areassociated with a specific disease. Expression profiles for variousdiseases are available, for example, for colon cancer (Llarena (2009) J.Clin. Oncol. 25:155 (e22182), ovarian cancer (Spentzos (2005) J. Clin.Oncol. 23:7911-7918), and lung adenocarcinoma (Takeuchi (2006) J. Clin.Oncol. 11:1679-1688). To give a similar example, what can also becharacterized is the influence of a released compound on mRNAsassociated with non-hepatic tumor cells that have metastasized to theliver (see, Barshack, Rosenwald, Bronfeld (2008) J. Clin. Oncol. 26:15Suppl. 11026, Barshack (2010) Int. J. Biochem. Cell Biol.42:1355-1362.).

Capturing the Transcriptome.

Methods are available for capturing mRNA by hybridizing their polyAgroup to immobilized poly(dT) (see, Dubiley (1997) Nucleic Acids Res.25:2259-2265; Hamaguchi, Aso, Shimada (1998) Clinical Chem.44:2256-2263; D. S. Hage (2005) Handbook of Affinity Chromatography,2^(nd) ed, CRC Press, page 549).

After capture of mRNA molecules released from the lysed cell (or cells),the bead-bound polynucleotide serves as a primer that supports reversetranscription from the mRNA, resulting in a bead-bound complementary DNA(cDNA), and where this bead-bound cDNA can be sequenced. Alternatively,the bead-bound cDNA can be released from the bead, where the bead-bound“response capture element” is coupled to the bead with a cleavablelinker, such as with a photocleavable linker. If a photocleavable linkeris used, cleaving conditions for releasing bead-bound compounds(compounds made from a chemical monomer library) but not also cleave thebead-bound “response capture element.”

Where cells are exposed to a bead-bound compound or to a compoundreleased from a bead, cells can be screened for a genetic response, forexample, by characterizing any changes in the transcriptome with orwithout exposure to the compound. Also, cells can be screened for aphenotypic response, for example, apoptosis, change in activity of oneor more cell-signaling proteins, or change in cell-surface expression ofone or more CD proteins. CD is Cluster of Differentiation (See, Lal(2009) Mol. Cell Proteomics. 8:799-804; Belov (2001) Cancer Res.61:4483-4489; IUIS/WHO Subcommittee on CD Nomenclature (1994) Bull.World Health Org. 72:807-808; IUIS-WHO Nobenclature Subcommittee (1984)Bull. World Health Org. 62:809-811). For some phenotypic responseassays, the cells must not be lysed.

The present disclosure addresses the unmet need to partition differentdrugs to different cells, for example, by exposing a single cell to onetype of drug where exposure occurs in a picowell.

The present disclosure also eliminates the need to prepare barcodedmRNA, where mRNA is released from a cell followed by preparing cDNA (inthis type of barcode, all mRNA from a given cell receives the samebarcode, when the transcriptosome is coverted to corresponding libraryof cDNA).

Parameters During Cell Incubation with the Perturbant.

For any given compound or some other type of perturbant, parameters thatcan be varied or controlled light, temperature, pH of cell medium,sound, concentration and exposure time to a reagent (reagent can be thecompound released from the bead, an enzyme substrate, a cytokine, acompound that is already an established drug, a salt), mechanicagitation, an antibody against a cell-surface protein, and so on.

Barcoding the Cell.

Cells can be incubated with a bead-bound compound or with the compoundfollowing cleavage from a bead-bound cleavable linker. During or afterincubation, cells can be barcoded with a membrane-bound barcode thatidentifies the purturbant. This membrane-bound barcode can be coupled tooligosaccharides of the cell membrane, polypeptides of the cellmembrane, or phospholipids of the cell membrane.

Response Capture Elements Other than Poly(dT).

Messenger RNA can be captured by way of the 5-prime 7-methylguanosinecap. This method is especially useful where there polyA tail is short(see, Blower, Jambhekar (2013) PLOS One. 8:e77700). Also, mRNA can becaptured using immobilized DNA that is specific for a coding region ofthe mRNA. This method is called, “RNA exome capture,” and variations ofthis name. According to Cieslik et al, “Unique to capturetranscriptomics is an overnight capture reaction (RNA-DNA hybridization)using exon-targeting RNA probes” (Cieslik (2015) Genome Res.25:1372-1381).

MicroRNA (miRNA).

The present disclosure can assess the influence of a released bead-boundcompound on expression profile of miRNAs in a given cell or,alternatively, on expression profiles of the population of mRNAs thatare specifically bound by a given species of miRNA (Jain, Ghosh, Barh(2015) Scientific Reports. 5:12832). For example, the present disclosureprovides a bead that contains: (1) Bead-bound compound; (2) Bead-boundDNA barcode; and (3) Bead-bound response capture element, where theresponse capture element either captures miRNA or where the responsecapture element includes a species of miRNA (as part of the responsecapture element). Expression profiles for microRNA have been found forvarious types of cancer, for example, breast cancer breast cancer (Tanja(2009) J. Clin. Oncol. 27:15 Suppl. 538).

Methods are available for capturing selected populations of mRNA fromthe entire transcriptome. Selectivity can be conferred by using one typeof microRNA, such as miR-34a, as a bridging compound in a “pull-down”assay. In brief, “The transcripts pulled down with miR-34a were . . .enriched for their roles in growth factor signaling and cell cycleprogression” (Lal, Thomas, Lieberman (2011) PLOS Genetics. 7:e1002363).The mRNA molecules that are captured are those that bind to the miR-34A.

Further methods for capturing mRNA and analyzing expression level isavailable (Bacher (2016) Genome Biology. 17:63; Svensson (2017) NatureMethods. 14:381; Miao and Zhang (2016) Quantitative Biol. 4:243; Gardini(2017) Nature Methods. 12:443). Cellular response taking the form ofchanges in enhancer RNA can be measured (see, Rahman (2017) Nucleic AcidRes. 45:3017).

The present invention is not to be limited by compositions, reagents,methods, systems, diagnostics, laboratory data, and the like, of thepresent disclosure. Also, the present invention is to not be limited byany preferred embodiments that are disclosed herein.

What is claimed is:
 1. A system for screening chemical compounds fortheir ability to modulate the biological activity of a cell or acomponent of a cell, comprising: a) an array comprising a plurality ofconfined volumes wherein each confined volume is separated from otherconfined volumes, b) a plurality of beads where each bead is suitablefor disposal in each confined volume, wherein each bead comprises aplurality of substantially identical bead-bound compounds, wherein saidbead-bound compounds are covalently linked to the bead by a cleavablelinker such that said compounds are releasable from said bead in ameasurable dose dependent manner as part of an assay, c) said beadsfurther comprising a plurality of substantially identical bead-bound DNAbarcodes linked to the bead (i) by a cleavable linker or (ii) by anon-cleavable linker, wherein if the DNA barcodes are linked to the beadby a cleavable linker, the cleavable linker is orthogonal to thecleavable linker used to link the bead-bound compounds to the bead, andwherein the DNA barcode identifies the compound.
 2. The system of claim1, wherein, when the bead-bound compounds are released from the bead,the bead-bound DNA barcodes are not released from the bead.
 3. Thesystem of claim 1, wherein each of the confined volumes comprises asingle bead.
 4. The system of claim 3, wherein the confined volume iseither a picowell or a droplet.
 5. The system of claim 1, wherein thecleavable covalent linker is cleaved by light, temperature change, pHchange, sound, salt or a change in oxidation.
 6. The system of claim 5,wherein the cleavable linker is cleaved by light.
 7. The system of claim6, wherein the light is ultraviolet light.
 8. The system of claim 1,wherein the component of a cell is a lipid, a protein, a carbohydrate,or a nucleic acid.
 9. The system of claim 8, wherein the component of acell is a nucleic acid.
 10. The system of claim 9, wherein the nucleicacid is mRNA.
 11. The system of claim 1, wherein the component of a cellis a cytokine, an antigen, or an enzyme.
 12. The system of claim 1,wherein the amount of compound released from the bead in a measurabledose dependent manner for testing is measured by a change in signalgenerated from a signal generating compound bound to a bead and distinctfrom the compound to be tested, wherein the signal generating compoundgenerates no signal or an attenuated signal on the bead and is capableof generating an increased signal once released from the bead, whereinthe increase in signal correlates to the amount of the to be testedcompound released from the bead, wherein the compound released from thebead and the signal generating compound released from the bead arereleased under the same conditions.
 13. The system of claim 12, whereinthe compound and the signal generating compound are bound to the samebead.
 14. The system of claim 13, wherein the signal generating compoundis a fluorescent compound whose fluorescence is quenched by a quencherwhen both are bond to the bead.
 15. The system of claim 1, wherein atleast one confined volume comprises a bead with bead-bound DNA barcodesand bead-bound compounds that are different from the bead-bound DNAbarcodes and bead-bound compounds on a bead in another confined volume.16. The system of claim 1, wherein the DNA barcode is linked to a beadby a cleavable linker and wherein the cleavable linker is different fromthe cleavable linker linking the bead-bound compound to the bead. 17.The system of claim 1, wherein the DNA barcode is linked to the bead bya non-cleavable linker.
 18. The system of claim 1, wherein the DNAbarcode is linked to the bead by a cleavable linker and wherein thecleavable linker is cleaved by a different mechanism than the cleavablelinker linking the bead-bound compound to the bead.